Design of Novel Melatonin Analogs for the Reduction of...

Design of Novel Melatonin Analogs for the Reduction ofIntraocular Pressure in Normotensive Rabbits

Pilar Alarma-Estrany, Ana Guzman-Aranguez, Fernando Huete, Assumpta Peral,Robert Plourde, Jr., Teresa Pelaez, Benjamin Yerxa, and Jesus PintorDepartamento Bioquimica (P.A.-E., A.G.-A., F.H., T.P., J.P.) and Optica (A.P.), Escuela Universitaria de Optica, UniversidadComplutense de Madrid, Madrid, Spain; and Inspire Pharmaceuticals, Inc., Durham, North Carolina (R.P., B.Y.)

Received December 16, 2010; accepted March 1, 2011

ABSTRACTMelatonin, the MT2 melatonin receptor agonist IIK7 [N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine], and theputative MT3 melatonin receptor agonist 5-MCA-NAT [5-methoxycarbonylamino-N-acetyltryptamine] have previouslybeen shown to reduce intraocular pressure (IOP) in ocular normo-tensive rabbits. To gain a better understanding of the structure-activity relationship of compounds that activate MT2 and MT3receptors mediating reductions in IOP, novel melatonin analogswith rationally varied substitutions were synthesized and tested fortheir effects on IOP in ocular normotensive rabbits (n � 160). Allsynthesized melatonin analogs reduced IOP. The best-effect low-ering IOP was obtained with the analogs INS48848 [methyl-1-methylene-2,3,4,9-tetrahydro-1H-carbazol-6-ylcarbamate],INS48862 [methyl-2-bromo-3-(2-ethanamidoethyl)-1H-indol-5-ylcarbamate], and INS48852 [(E)-N-(2-(5-methoxy-1H-indol-3-

yl)ethyl)-3-phenylprop-2-enamide]. These compounds produceddose-dependent decreases in IOP that were maximal at 0.1 mM(total dose of 0.259 �g for INS48848, 0.354 �g for INS48862, and0.320 �g for INS48852) and 1 mM (total dose of 2.59 �g forINS48848, 3.54 �g for INS48862, and 3.20 �g for INS48852), withmaximal reductions of 36.0 � 4.0, 24.0 � 1.5, and 30.0 � 1.5% forINS48848, INS48862, and INS48852, respectively. Studies usingmelatonin receptor antagonists (luzindole, prazosin, and DH97[N-pentanoyl-2-benzyltryptamine]) indicated that INS48862 andINS48852 activate preferentially a MT2 melatonin receptor andsuggest that INS48848 may act mainly via a MT3 receptor. Themost effective compounds were also well tolerated in a battery ofstandard ocular surface irritation studies. The implication of thesefindings to the design of novel drugs to treat ocular hypertensionis discussed.

IntroductionGlaucoma is a group of diseases characterized by retinal and

optic neuropathy and progressive visual field loss. The mostprevalent type, open angle glaucoma, is estimated to account forapproximately 15% blindness worldwide (Thylefors and Negrel,

1994). The pathology of this type is secondary to elevated intra-ocular pressure (IOP), and reduction of IOP is the most commontreatment modality.

In the normal eye, a balance of formation and outflow ofaqueous humor regulates IOP, maintaining a mean IOP ofapproximately 16 mm Hg. In open angle glaucoma, normalaqueous humor outflow through the trabecular meshwork isimpeded, and there is a consequential rise in IOP to valueshigher than 21 mm Hg (Schottenstein, 1996).

Circadian fluctuation of IOP is well established, and therelationship between melatonin and IOP has been explored inview of the involvement of pineal melatonin in the regulation of

This work was supported by grants from Ministerio de Ciencia e Inno-vacion [SAF2007-60835, SAF2010-16024]; RETIC Red de Patología Oculardel Envejecimiento, Calidad Visual y Calidad de Vida [RD07/0062/0004];NEUROTRANS CM [S-SAL 0253-2006]; and BSCH-UCM [GR58/08].

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.110.178319.

ABBREVIATIONS: IOP, intraocular pressure; 5-MCA-NAT, 5-methoxycarbonylamino-N-acetyltryptamine; IIK7, N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine; DH97, N-pentanoyl-2-benzyltryptamine; EA, electron affinity; FOSA, hydrophobic solvent-accessiblesurface area; DMSO, dimethyl sulfoxide; INS48848, methyl-1-methylene-2,3,4,9-tetrahydro-1H-carbazol-6-ylcarbamate; INS48862, methyl-2-bromo-3-(2-ethanamidoethyl)-1H-indol-5-ylcarbamate; INS48852, (E)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-3-phenylprop-2-enamide; INS48864,S-(butylperoxy)-N-((5-methoxy-1H-indol-3-yl)methyl)thiohydroxylamine; INS48879, diphenyl (5-methoxy-1H-indol-3-yl)methylphosphoramidate;INS48476, methyl-3-(propanamidomethyl)-1H-indol-5-ylcarbamate; INS48793, methyl-2-bromo-3-(ethanamidomethyl)-1H-indol-5-ylcarbamate;INS48834, methyl-3-(ethanamidomethyl)-2-methyl-1H-indol-5-ylcarbamate; INS48497, allyl-3-(ethanamidomethyl)-1H-indol-5-ylcarbamate;INS48838, N-((5-methoxy-1H-indol-3-yl)methyl)propanamide; INS48853, N-((5-methoxy-1H-indol-3-yl)methyl)-2-methylpropanamide; INS48836,N-((5-methoxy-1H-indol-3-yl)methyl)benzamide; INS48882, 1-((5-methoxy-1H-indol-3-yl)methyl)-6-oxopiperidine-2-carboxylic acid; INS48887,methyl-3-(ethanamidomethyl)-1H-indol-5-ylcarbamate; INS48863, N-((5-methoxy-1H-indol-3-yl)methyl)methanesulfonamide; INS48803, methyl-3-(2-ethanamidoethyl)-1-methyl-1H-indol-5-yl(methyl)carbamate.

0022-3565/11/3373-703–709$25.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 337, No. 3Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 178319/3687322JPET 337:703–709, 2011 Printed in U.S.A.

703

at ASPE

T Journals on O

ctober 9, 2018jpet.aspetjournals.org

Dow

nloaded from

http://jpet.aspetjournals.org/

many circadian rhythms (Moore, 1997; Lewy, 1999). Despitecontradictory findings, the most widely held conclusion is thatdecreased IOP correlates positively with increased intraocularmelatonin levels (Rohde et al., 1985; Komaromy et al., 1998;Pointer, 1997). However, pharmacological characterization ofmelatonin receptors reveals three receptor subtypes (MT1, MT2,and MT3; Dubocovich, 1995) and that the relationship betweenindividual receptor subtypes and IOP has not been evaluatedcomprehensively. The selective MT3 receptor ligand 5-methoxy-carbonylamino-N-acetyltryptamine (5-MCA-NAT) has beenshown to be a potent ocular hypotensive agent (Pintor et al.,2001, 2003; Serle et al., 2004), and the selective MT2 receptoragonist N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine (IIK7; Sugden et al., 1999) also markedlydecrease the IOP and was inhibited by selective MT2 receptorantagonists (Alarma-Estrany et al., 2008). Both 5-MCA-NATand IIK7 offer themselves as starting points for new classes ofdrugs to lower IOP and for treating ocular hypertension andglaucoma.

With the 5-MCA-NAT and IIK7 findings as a startingpoint, we prepared a series of 16 compounds to evaluate thestructure-activity relationships that determine the ability ofthese compounds to lower IOP. The compounds were testedfor their ability to lower IOP in ocular normotensive rabbits,and molecular features associated with enhanced efficacywere determined. Furthermore, as a preliminary assessmentof safety, ocular surface tolerance tests were carried out withthe most efficacious compounds.

Materials and MethodsChemistry. Compounds INS48476, INS48497, INS48834, and

INS48887 were synthesized using a general procedure developedby Macor et al. (1993). In brief (Fig. 1A), an appropriately substi-tuted 5-nitroindole was treated with oxalylchloride followed byammonia and then reduced with borane to produce the tryptaminederivative. Acylation was effected with an appropriate anhydrideor acid chloride. The nitro group then was reduced and acylatedwith an appropriate chloroformate. Compound INS48862 wasformed by site-specific bromination of the parent compound usingN-bromosuccinimide/acetic acid.

Compounds INS48803 and INS48862 were obtained by alkylationof precursor acyltryptamines with NaH/methyl iodide/dimethyl for-mamide and NaH/benzyl bromide/N,N-dimethylformamide, respec-tively, whereas compound INS48848 was obtained by hydrogenationand acylation of the parent nitrotetrahydrocarboline. Melatonin an-alogs (Fig. 1B) INS48836 and INS48852 were prepared by treatmentof 5-methoxytryptamine with the appropriate acid chloride and tri-ethylamine in dichloromethane, whereas INS48838, INS48853, andINS48882 were prepared by treatment of 5-methoxytryptamine withthe appropriate anhydride and triethylamine in dichloromethane.Compounds INS48863 and INS48864 were formed by treatment of5-methoxytryptamine with the corresponding sulfonyl chlorides andtriethylamine in dichloromethane. Compound INS48879 was synthe-sized by treatment of 5-methoxytryptamine with diphenylphospho-ryl chloride and triethylamine in dichloromethane. Computation ofADME (absorption, distribution, metabolism, and excretion) andmolecular properties was carried out in QikProp (version 1.6; Sch-rodinger, New York, NY) and visualized using Spotfire 7 (Somerville,

Fig. 1. Scheme of the synthesis of the novel melatoninanalogs.

704 Alarma-Estrany et al.

at ASPE

T Journals on O


Dow

nloaded from


MA). All compounds exhibited satisfactory 1H NMR and high-reso-lution mass spectra.

Animals. Normotensive New Zealand white rabbits (160), weigh-ing 3 � 0.5 kg, were kept in individual cages with food and water adlibitum. They were maintained under a controlled 12-h/12-h light/dark cycle in the School of Optics’ animal facilities (UniversidadComplutense de Madrid Animal House). All of the procedures com-plied with the Association for Research in Vision and OphthalmologyStatement for the Use of Animals in Ophthalmology and VisionResearch and also are in accordance with the European Communi-ties Council Directive (86/609/EEC).

Formulation and Method of Administration. All compoundswere formulated in isotonic saline containing 1% dimethyl sulfoxide(DMSO) and tested at a final concentration of 0.648 �g for INS48848,0.885 �g for INS48862, 0.801 �g for INS48852, 0.811 �g forINS48864, 1.056 �g for INS48879, 0.723 �g for INS48476, 0.885 �gfor INS48793, 0.723 �g for INS48834, 0.813 �g for INS48497, 0.616�g for INS48838, 0.651 �g for INS48853, 0.736 �g for INS48836,1.142 �g for INS48882, 0.728 �g for INS48887, 0.705 �g forINS48863, and 0.758 �g for INS48803 (all compounds of 0.25 mM,giving a dose of 2.5 nmol in a volume of 10 �l). Compounds wereapplied unilaterally to the cornea at a fixed volume of 10 �l. Thecontralateral eye received the same volume of vehicle (1% DMSO v/v,0.9% w/v saline). Because the application of the tonometer mayproduce discomfort in the rabbits, corneas were anesthetized byapplying 10 �l of oxibuprocaine/tetracaine (1:4; Colicursi DoubleAnaesthetic; Alcon Cusi, Barcelona, Spain) before IOP was measured.

Experiments were performing following a blinded design; no indi-cation was given to the experimenter with regard to the appliedsolution (agent or vehicle). IOP measurements were made using aTonopen aplanation contact tonometer before and at several timesafter instillation of a compound. IOP was followed up to 5 h to studythe time course of the effect. Afterward, the three most active com-pounds at decreasing IOP were tested again to study the time courseof up to 8 h at a total dose of 0.259 �g for INS48848, 0.320 �g forINS48852, and 0.354 �g for INS48862 (0.1 mM final concentration,giving a dose of 1 nmol in a volume of 10 �l). To know whichmelatonin receptors were being activated, substances that producedhypotension were tested in the presence of N-pentanoyl-2-benzyl-tryptamine (DH97), prazosin, or luzindole. Ten microliters of theseantagonists were added 30 min before the application of either mel-atonin analog at a dose of 100 �g (29.9 mM DH97, 23.82 mMprazosin, and 34.20 mM luzindole). Furthermore, dose-response ex-periments were carried out for the three most active compounds. Onany given day, only a single dose was tested on a single animal,which was washed out at least 2 days between doses.

To examine ocular surface short-term tolerance, 50 �l (0.25 mM) ofeach formulated compound was instilled onto the cornea of both eyesin three animals, and observations were made using the Draize scale(Draize et al., 1944) immediately before treatment and at 2, 5, 15,and 60 min after treatment. The Draize scale is a long-establishedstandard for assessing ocular surface tolerability and involves scor-ing six components of the eye’s anterior segment: conjunctival che-mosis, discharge and redness; corneal opacity and involvement; andiritis.

ResultsStructure-Activity Relationships and ADME Stud-

ies. Table 1 displays the structure of each compound alongwith its maximal IOP-lowering effect during the 5-h period ofexamination. In general, compounds in which R4 was analkoxycarbonylamino group were equally or slightly moreefficacious than those in which R4 was a methoxy group,followed in efficacy by �OMe derivatives. INS48848,INS48852, and INS48862 showed some differences in quan-titative calculation of ADME and molecular properties in

QikProp. LogBB (which is a measure of the partitioningbetween brain tissues and the blood) of the selected effica-cious compounds was relatively poor and clustered (range�1.3 to �0.8) relative to the broad set (range �1.5 to �0.4).A unique relationship was identified between the hydropho-bic solvent-accessible surface area (FOSA), the electron af-finity (EA), and the dipole moment of the three most effica-cious compounds (Fig. 2). All three are set apart from themajority of the set with substantially higher EAs (mean “0.3eV versus mean” 0 eV for the complete array), lower dipolemoments (mean “4.7 versus mean” 6.5 for the complete ar-ray), and somewhat lower FOSA (mean “225 Å3 versus mean”290 Å3 for the complete array).

Dose-Response and Time Course Experiments. All ofthe synthesized compounds exerted a modulatory effect onIOP as can be seen in Table 1. INS48848, INS48862, andINS48852, which evoked the greatest reductions in IOP at atotal dose of 0.648 �g for INS48848, 0.885 �g for INS48862,and 0.801 �g for INS48852 (0.25 mM, giving a dose of 2.5nmol in a volume 10 �l), produced dose-dependent decreasesin IOP, which were maximal at 0.1 mM (total dose of 0.259 �gfor INS48848, 0.354 �g for INS48862, and 0.320 �g forINS48852), �1 mM (total dose of 2.59 �g for INS48848, 3.54�g for INS48862, and 3.20 �g for INS48852) (1–10 nmol, 10�l), with maximal reductions of 36.0 � 4.0, 24.0 � 1.5, and30.0 � 1.5% for INS48848, INS48862, and INS48852, respec-tively (n � 8; Fig. 3). The analysis of the curves permitted thecalculation of the pD2 values (�logEC50) of 5.5 � 0.2, 5.7 �0.3, and 5.5 � 0.2 for INS48848, INS48862, and INS48852,respectively. These values are equivalent to doses of 8.19 ngfor INS48848, 7.04 ng for INS48862, and 10.12 ng forINS48852.

The time course of the changes in IOP induced by themelatonin analogs was also examined. The three compounds,administered at a single dose of 0.259 �g for INS48848, 0.354�g for INS48862, and 0.320 �g for INS48852 (0.1 mM, givinga dose of 1 nmol in a volume of 10 �l), evoked a reduction ofthe IOP, with a maximal effect observable between 30 minand 2 h after application (Fig. 4). In particular, and in agree-ment with the concentration-response studies, the melatoninanalog INS48848 reduced IOP by 35% at 1 h, INS48852produced a 31% reduction at 2 h, and INS48862 decreasedIOP by 24% at 30 min. For all of these compounds, IOPreturned to its normal level within 8 h of the instillation (n � 8).

Effects of Melatonin Receptor Antagonists. Pretreat-ment with the nonspecific melatonin receptor antagonist luz-indole (100 �g, 30 min) abolished the ocular hypotensiveeffect of INS48848 (0.259 �g) (1 nmol/10 �l) but did notinhibit the effect of the INS48862 or INS48852. Pretreatmentwith the MT3 binding site antagonist, prazosin, abolished theocular hypotensive effect of INS48848 (0.259 �g) (1 nmol/10�l) but did not inhibit the actions of INS48862 (0.354 �g) orINS48852 (0.320 �g) (both at 1 nmol/10 �l). The MT2 antag-onist DH97 (100 �g equivalent to 29.9 mM) inhibited theeffects of INS48862 and INS48852 but had not effectedagainst INS48848 (Fig. 5). We extended the experimentswith this MT2 melatonin receptor antagonist by assaying itat graded concentrations from 0 to 100 �g (Fig. 6). Theseexperiments confirmed that INS48862 and INS48852 wereantagonized by DH97 but not INS48848. These results indi-cate that INS48862 and INS48852 activate preferentially a

Melatonin Analogs Reduce IOP 705

at ASPE

T Journals on O


Dow

nloaded from


MT2 melatonin receptor and suggest that INS48848 may actmainly via a MT3 receptor.

Tolerance Test. By the criteria, of conjunctival chemosis,discharge, and redness; corneal opacity and involvement; andiritis in the Draize scale, all of the compounds tested werewell tolerated (results not shown).

DiscussionIn the present experimental work, we describe the synthe-

sis of new melatonin analogs that reduce IOP in normal NewZealand White rabbits. Because there are substantial differ-ences between human and rabbit ocular anatomy and phys-iology (Bito, 1984), the screening of the best compounds wasbased on maximal IOP-lowering effect rather than on theeffect at a selected time point.

The outstanding feature associated with high efficacy ap-pears to be structural rigidity and increased hydrophobicityon the eastern hemisphere of the molecule, as exemplified byINS48848, INS48852, and INS48862. In the case of com-pounds INS48879 and INS48864, the positive effect of addedhydrophobicity may be mitigated by the higher charge den-sity of the phosphoryl and sulfonyl groups compared with anacyl group.

Substitution of the N-acetyl group of the 3-position sidechain with a charged functional group or a highly polarizedgroup (INS48863, INS48882, and INS48887) almost abol-ished IOP-lowering activity, as did methylation of the R4-carbamate (INS48803), suggesting that hydrogen bondingability at R4 may be a critical point for activity. However,quantitative calculation of ADME and molecular properties

TABLE 1Structure of novel melatonin analogs next to its maximal IOP-lowering effect during the 5-h period of examination

Compound R1 R2 R3 R4 Maximal Percentage IOP Reduction

INS48848 H NHCO(O)Me 36

INS48852 H H OMe 33

INS48862 Bn H NHCO(O)Me 26

INS48864 H H OMe 25

INS48879 H H OMe 22

INS48476 H H NHC(O)OMe 22

INS48793 H Br NHC(O)OMe 22

INS48834 H Me NHC(O)OMe 20

INS48497 H H 20

INS48838 H H OMe 20

INS48853 H H OMe 20

INS48836 H H OMe 18

INS48882 H H OMe 13

INS48887 H H NHC(O)OMe 11

INS48863 H H OMe 10

INS48803 Me H NMeC(O)OMe 8


at ASPE

T Journals on O


Dow

nloaded from


in QikProp revealed significant disparity between INS48848and the other two most efficacious compounds (INS48852and INS48862). Although it is tempting to speculate aboutdifferences in binding mode, based upon this information, itis also important to understand that because these com-pounds are administered onto the ocular surface, more spe-cific ADME properties, such as Topliss’ corneal permeability(Yoshida and Topliss, 1996), could be more relevant. Exam-ination of calculated molecular properties was somewhatmore enlightening. Thus, we observed a unique relationshipbetween the FOSA, EA, and the dipole moment of the threemost efficacious compounds.

Using a 0.1 mM concentration of INS48848 (total dose0.259 �g), INS48852 (total dose 0.320 �g), and INS48862(total dose 0.354 �g) to easily compare with concentrations ofapplied melatonin, 5-MCA-NAT, and IIK7 in recent studies(Alarma-Estrany et al., 2007, 2008, 2009), responses weresimilar to those obtained using 0.25 mM. Moreover, theseFig. 2. Computation of ADME and molecular properties of the novel

melatonin analogs.

Fig. 3. Effects of the three most efficacious novel melatoninanalogs on rabbit IOP. Top panels, structure of the threemost efficacious novel melatonin analogs. Bottom panel,dose-response curves for INS48848, INS48862, andINS48852 melatonin analogs where tested doses were be-tween 10 mM (total dose of 25.92 �g for INS48848, 35.42 �gfor INS48862, and 32.04 �g for INS48852) and 1 nM (totaldose of 0.002592 ng for INS48848, 0.003542 ng forINS48862, and 0.003204 ng for INS48852) (n � 8 for eachcompound). One hundred percent represents the IOP be-fore application of any drug. Values represent the mean �S.E.M. of eight independent experiments.


at ASPE

T Journals on O


Dow

nloaded from


compounds decreased IOP in a dose-dependent manner sim-ilar to melatonin, 5-MCA-NAT, and IIK7 (Pintor et al., 2001;Alarma-Estrany et al., 2008), confirming the efficiency ofthese melatonin analogs for decreasing IOP in a way similarto 5-MCA-NAT and IIK7.

The effects of INS48848 were completely blocked by pra-zosin, an antagonist of MT3 melatonin receptors (Paul et al.,1999; Pintor et al., 2003; Xia et al., 2008), and were potentlyinhibited by luzindole, a nonselective antagonist of melatoninreceptors (Pintor et al., 2003). However, DH97, an MT2 re-ceptor antagonist (Chen et al., 2005; Alarma-Estrany et al.,2007; Mendoza-Vargas et al., 2009), had little effect againstINS48848. In sharp contrast, the results obtained forINS48862 and INS48852 were the opposite. Luzindole andprazosin had no significant effects against these two com-pounds, whereas DH97 blocked them completely. These re-sults strongly suggest that the compound INS48848 could beacting through the MT3 melatonin receptors and that thecompounds INS48862 and INS48852 could be acting prefer-entially through MT2 melatonin receptors.

Taking account that prazosin is also a �1-adrenoceptorantagonist, it should be considered that INS48848 could havebeen acting through adrenoceptors instead of melatonin re-ceptors. However, this remains to be investigated in detail;5-MCA-NAT, which is similarly antagonized by prazosin, isnot antagonized by the �1-selective adrenoceptor antagonistcorynanthine (Pintor et al., 2003).

Considering that the compounds described here can acti-vate MT2 and MT3 receptors, an interesting approach couldbe to combine them to get stronger reductions in IOP. Ac-cording to our results, a combination of INS48852 (whichproduces 30% IOP reduction via MT2 receptors) plusINS48848 (40% reduction via MT3) could be an interestingone. Nevertheless, it would be necessary to optimize the

concentrations and instillation volumes of both compounds toget the most effective formulation.

Designing and developing new drugs to treat glaucomarequires consideration of many factors, including efficacy,specificity, bioavailability, safety, and toxicity. Our presentstudy has begun to probe the viability of a new class ofcompounds as a source for IOP-lowering drugs. With these

Fig. 4. Time course of the changes in IOP in response to melatoninanalogs instillation. Effects of INS48848, INS48862, and INS48852 mel-atonin analogs at total dose of 0.259 �g for INS48848, 0.354 �g forINS48862, and 0.320 �g for INS48852 (0.1 mM, 10 �l) on IOP werefollowed for 8 h (n � 8 for each compound). One hundred percent repre-sents the IOP before application of any drug. Values represent themean � S.E.M. of eight independent experiments. ��, p � 0.01 versussaline; ��, p � 0.001 versus vehicle; �, p � 0.01 versus vehicle.

Fig. 5. Antagonism by prazosin, luzindole, and DH97 of the responsesproduced by INS48848 (total dose of 0.259 �g), INS48862 (total dose of0.354 �g), and INS48852 (total dose of 0.320 �g) melatonin analogs (0.1mM, 10 �l, and n � 8 for all compounds). Values are the mean � S.E.M.of eight independent experiments. �, p � 0.01 versus melatonin analogalone; ��, p � 0.0001 versus melatonin analog alone.


at ASPE

T Journals on O


Dow

nloaded from


studies in rabbits, we begin to establish the relationship ofvaried molecular properties and substitution patterns withefficacy. Furthermore, we have determined that topical ap-plication of representative compounds from this class is welltolerated and does not cause short-term ocular surface irri-tation. In addition, we have recently found out that it ispossible to dissolve these compounds in solvents that areacceptable for use in topical applications for humans (An-dres-Guerrero et al., 2009), rather than in DMSO or ethanol,which are not. Therefore, these novel drugs have clinicalpotential to treat ocular hypertension and glaucoma.

Acknowledgments

We thank Dr. Charles H. V. Hoyle for critical reading of thismanuscript.

Authorship Contributions

Participated in research design: Yerxa and Pintor.Conducted experiments: Alarma-Estrany, Peral, Pelaez, Huete,

and Plourde.Contributed new reagents or analytic tools: Plourde.Performed data analysis: Guzman-Aranguez and Pintor.Wrote or contributed to the writing of the manuscript: Guzman-

Aranguez and Pintor.

ReferencesAlarma-Estrany P, Crooke A, Peral A, and Pintor J (2007) Requirement of intact

sympathetic transmission for the ocular hypotensive effects of melatonin and5-MCA-NAT. Auton Neurosci 137:63–66.

Alarma-Estrany P, Crooke A, Mediero A, Pelaez T, and Pintor J (2008) Sympatheticnervous system modulates the ocular hypotensive action of MT2-melatonin recep-tors in normotensive rabbits. J Pineal Res 45:468–475.

Alarma-Estrany P, Crooke A, and Pintor J (2009) 5-MCA-NAT does not act throughNQO2 to reduce intraocular pressure in New-Zealand white rabbit. J Pineal Res47:201–209.

Andres-Guerrero V, Alarma-Estrany P, Molina-Martínez IT, Peral A, Herrero-Vanrell R, and Pintor J (2009) Ophthalmic formulations of the intraocular hypo-tensive melatonin agent 5-MCA-NAT. Exp Eye Res 88:504–511.

Bito LZ (1984) Species differences in the responses of the eye to irritation andtrauma: a hypothesis of divergence in ocular defense mechanisms, and the choiceof experimental animals for eye research. Exp Eye Res 39:807–829.

Chen Y, Tjong YW, Ip SF, Tipoe GL, and Fung ML (2005) Melatonin enhances thehypoxic response of rat carotid body chemoreceptor. J Pineal Res 38:157–163.

Draize JH, Woodard G, and Calvery HO (1944) Methods for the study of irritationand toxicity of substances applied topically to the skin and mucous membranes.J Pharmacol Exp Ther 82:377–390.

Dubocovich ML (1995) Melatonin receptors: are there multiple subtypes? TrendsPharmacol Sci 16:50–56.

Komaromy AM, Brooks DE, Kubilis PS, Dawson WW, Sapp HL Jr, Nelson G, CollinsBR, and Sherwood MB (1998) Diurnal intraocular pressure curves in healthyrhesus macaques (Macaca mulatta) and rhesus macaques with normotensive andhypertensive primary open-angle glaucoma. J Glaucoma 7:128–131.

Lewy AJ (1999) Melatonin as a marker and phase-resetter of circadian rhythms inhumans. Adv Exp Med Biol 460:425–434.

Macor JE, Post R, and Ryan K (1993) A simple synthesis of 5-amino-3-(2-dimethylaminoethyl) indole[5-amino-N,N-dimethyl-tryptamine]. Synthetic Com-munications 23:65–72.

Mendoza-Vargas L, Solís-Chagoyan H, Benítez-King G, and Fuentes-Pardo B (2009)MT2-like melatonin receptor modulates amplitude receptor potential in visualcells of crayfish during a 24-hour cycle. Comp Biochem Physiol A Mol IntegrPhysiol 154:486–492.

Moore RY (1997) Circadian rhythms: basic neurobiology and clinical applications.Annu Rev Med 48:253–266.

Paul P, Lahaye C, Delagrange P, Nicolas JP, Canet E, and Boutin JA (1999)Characterization of 2-[125I]iodomelatonin binding sites in Syrian hamster periph-eral organs. J Pharmacol Exp Ther 290:334–340.

Pintor J, Martin L, Pelaez T, Hoyle CH, and Peral A (2001) Involvement of melatoninMT(3) receptors in the regulation of intraocular pressure in rabbits. Eur J Phar-macol 416:251–254.

Pintor J, Pelaez T, Hoyle CH, and Peral A (2003) Ocular hypotensive effects ofmelatonin receptor agonists in the rabbit: further evidence for an MT3 receptor.Br J Pharmacol 138:831–836.

Pointer JS (1997) The diurnal variation of intraocular pressure in non-glaucomatoussubjects: relevance in a clinical context. Ophthalmic Physiol Opt 17:456–465.

Rohde BH, McLaughlin MA, and Chiou LY (1985) Existence and role of endogenousocular melatonin. J Ocul Pharmacol 1:235–243.

Schottenstein EM (1996) Intraocular pressure and tonometry, in The Glaucomas,Basic Sciences (Ritch R, Shields MB, and Krupin T, eds), pp 407–408, CV Mosby,St. Louis, MO.

Serle JB, Wang RF, Peterson WM, Plourde R, and Yerxa BR (2004) Effect of5-MCA-NAT, a putative melatonin MT3 receptor agonist, on intraocular pressurein glaucomatous monkey eyes. J Glaucoma 13:385–388.

Sugden D, Yeh LK, and Teh MT (1999) Design of subtype selective melatoninreceptor agonists and antagonists. Reprod Nutr Dev 39:335–344.

Thylefors B and Negrel AD (1994) The global impact of glaucoma. Bull WHO72:323–326.

Xia CM, Shao CH, Xin L, Wang YR, Ding CN, Wang J, Shen LL, Li L, Cao YX, andZhu DN (2008) Effects of melatonin on blood pressure in stress-induced hyperten-sion in rats. Clin Exp Pharmacol Physiol 35:1258–1264.

Yoshida F and Topliss JG (1996) Unified model for the corneal permeability ofrelated and diverse compounds with respect to their physicochemical properties.J Pharm Sci 85:819–823.

Address correspondence to: Dr. Jesus Pintor, Departamento Bioquímica, Es-cuela Universitaria de Optica, Universidad Complutense Madrid, C/Arcos deJalon 118, 28037 Madrid, Spain. E-mail: [email protected]

Fig. 6. Effect of DH97 on responses to melatonin analogs. Graded dosesof DH97 together with INS48848 (total dose of 0.259 �g), INS48862 (totaldose of 0.354 �g), and INS48852 (total dose of 0.320 �g) were applied (n �8 for each compound). One hundred percent represents the IOP beforeapplication of any drug. Values represent the mean � S.E.M. of eightindependent experiments. �, p � 0.01 versus control; ��, p � 0.001 versuscontrol; ��, p � 0.0001 versus control.


at ASPE

T Journals on O


Dow

nloaded from


Design of Novel Melatonin Analogs for the Reduction of...

Documents

Transcript of Design of Novel Melatonin Analogs for the Reduction of...