European Journal of Pharmacology 567 (2007) 145–148www.elsevier.com/locate/ejphar

Short communication

Hypotensive effect of profilin on rabbit intraocular pressure

Miguel Morales a, Azucena Gómez-Cabrero a, Assumpta Peral b, Xavier Gasull a, Jesús Pintor c,⁎

a Laboratory of Neurophysiology, Department of Physiological Sciences I, Institute of Biomedical Investigations August Pi I Sunyer (IDIBAPS), School of Medicine,University of Barcelona, Casanova 143, E-08036 Barcelona, Spain

b Department of Optics II University Complutense, c/Arcos de Jalón s/n E-28037 Madrid, Spainc Department of Biochemistry, E.U. Optica, University Complutense, c/Arcos de Jalón s/n E-28037 Madrid, Spain

Received 19 December 2006; received in revised form 3 April 2007; accepted 16 April 2007Available online 22 April 2007

Abstract

Ocular hypertension is a negative process that occurs within the eye and is the main risk factor to develop glaucoma, a progressive loss ofvision due to degeneration of retinal ganglion cells. The protein transduction technique allows a cargo to cross biological membranes. Using thistechnique we have previously shown that a membrane permeable version of profilin I (PTD4-profilin) increased aqueous humour outflow facility.Here we have investigated if a topical application of PTD4-profilin was able to modify intraocular pressure in rabbits. 10 μM PTD4-profilin(10 μL), reduced intraocular pressure by 20% compared to the control vehicle, this value being in the range of other commercial drugs, whichproduced intraocular pressure reductions between 18 and 35%. The mean-time effect for PTD4-profilin was 6.8 h and was also in the same rangeas commercial products that provided values between 4.3 and 5.5 h. According to the results presented here we propose PTD4-profilin as a newapproach for the treatment of ocular hypertension and PTD4 as a new strategy to facilitate the penetration of molecules into the eye.© 2007 Elsevier B.V. All rights reserved.

Keywords: Glaucoma; IOP; Intraocular pressure; PTD4-profilin

1. Introduction

The search for new, effective and more selective compoundsfor the treatment of ocular hypertension and glaucoma isbringing molecules with structures far away for the classicalcommercial pharmaceutical compounds. The discovery of thesenew potential therapeutic agents contribute not only tounderstand the molecular mechanisms involved in the regula-tion of intraocular pressure, but also to offer new substanceswith hypotensive properties and with less side effects (Pintor,2005).

Compounds such as extracellular nucleotides or indole deri-vatives such as melatonin, increase the number of agents thatmay be useful for glaucoma treatment (Soto et al., 2005; Pintoret al., 2003). Nevertheless, either these new molecules, or otherwell established ones, produce their effects through two mainmechanisms: receptor agonism/antagonism or enzyme inhibi-

⁎ Corresponding author. Dep Bioquímica E.U. Optica UCM c/Arcos de Jalóns/n, E-28037 Madrid Spain. Tel.:+ 34 91 3946859; fax: +34 91 3946885.

E-mail address: jpintor@vet.ucm.es (J. Pintor).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.04.020

tion. Strategies are therefore to activate or block a receptor, suchas carbachol and timolol respectively, or to inhibit an enzyme,like in the case of dorzolamide (Uva et al., 2006).

Defining new mechanistic approaches for the reduction ofintraocular pressure different from the described above de-mands to integrate new biochemical findings and techniqueswith ocular physiology. Cytoskeleton dynamics have been im-plicated in trabecular meshwork function and aqueous humourregulation since latrunculins and other actin-depolymerizingdrugs increased outflow facility (Peterson et al., 1999; Petersonet al., 2000a,b). Moreover, the Rho/Rho-kinase pathway reor-ganizes the actomyosin cytoskeleton inducing trabecular mesh-work contractility that finally regulates aqueous humour outflowfacility (Rao et al., 2005).

One of these proteins capable to reorganize the actin cyto-skeleton is profilin. Profilin is a small (12–15 kDa) proteinabundantly expressed in all eukaryotic cells that bind actinmonomers catalyzing the exchange of ADP to ATP-actin pro-moting actin filaments polymerization (Wolven et al., 2000)thereby, contributing to the regulation of actin dynamics (Witke,2004).

Fig. 1. Time-course of PTD4-profilin induced changes in intraocular pressureafter a single instillation. A single dose of PTD4-profilin (10 μM, 10 μL)produced a decrease in intraocular pressure (from 22.0±0.6 to 18.1±0.9 mmHg) that remained below the control value for more than 5 h (solid squares).Application of PTD4 alone (PTD4-HA; open squares) or profilin alone (opentriangles) did not modify intraocular pressure when compared to vehicle (saline,open circles). Values represent the mean±S.E.M. of six independent experi-ments. ⁎Pb0.05 versus the effect of vehicle alone.

146 M. Morales et al. / European Journal of Pharmacology 567 (2007) 145–148

Recently a novel approach for delivery of proteins and othersubstances across biological membranes and tissues has emerged;protein transduction domains (PTDs) derived from the TAT familycan carry a protein across the cell membrane into the cytoplasmin a receptor-independent fashion (Schwarze et al., 1999). In aprevious report, we have demonstrated that a recombinant proteinobtained by fusing profilin I to the PTD4 transduction domain, amore efficient version of TAT (Ho et al., 2001), was able to crossthe trabecular meshwork cell membranes in culture and whenperfused into bovine anterior segments increased aqueous humouroutflow facility (Gomez-Cabrero et al., 2005).

In the present work, we move forward describing the effectof PTD4-profilin on intraocular pressure in New Zealand rab-bits and how this effect is comparable in magnitude with thatproduced by other commercial anti-glaucoma drugs.

2. Materials and methods

2.1. Animals

New Zealand white rabbits, weighting 2–2.5 kg, were kept inindividual cages with ad libitum food and water. They weresubmitted to controlled 12 h/12 h light/dark cycles. All theprotocols herein comply with the ARVO Statement for the Useof Animals in Ophthalmology and Vision Research and also arein accordance with the European Communities CouncilDirective (86/609/EEC).

2.2. Recombinant profilin cloning and purification

cDNA human profilin sequence was cloned into a modifiedversion of the pRSETA expression vector (for details see Gomez-Cabrero et al., 2005). The vector was previously created to allowthe generation of recombinant proteins fused to the PTD4 (proteintransduction domain 4, sequence; YARAAARQARA; Ho et al.,2001). Human profilin I was generously provided by Drs. HitomiMimuro and Tadaomi Takenawa, University of Tokyo, Japan.Protein expression and purification was carried out as describedpreviously (Gomez-Cabrero et al., 2005). Briefly, pellets fromBL21-PLys bacteria were isolated and lysated. The recombinantprotein was purified using a Ni-NTA resin (Quiagen, Hilden,GmgH) and eluted with imidazol. Buffer exchange and con-centration of eluted proteins was performed by centrifugation inAmicon Ultra-15 10000 MWCO centrifugal filters (MilliporeIberica, Madrid, Spain). Proteins were frozen in liquid N2 andstored at − 80°C in 10–15% glycerol–PBS. PTD4-HA, a hema-toglutinin domain fused to the transduction domain (sequence:YARAAARQARAGEQKLISEEDL) was purchased from Gen-Script (Piscataway, NJ USA).

2.3. Formulation and method of administration

PTD4-profilin was formulated in isotonic saline and tested ata final concentration of 10 μM. The compound was appliedunilaterally to the cornea at a fixed volume of 10 μL. Thecontralateral eye received the same volume of saline (NaCl 0.9%,vehicle). In order to test whether PTD4-HA or profilin alone

produces any effect on intraocular pressure (IOP), both wereassayed in the same fashion as PTD4-profilin (10 μM, 10 μL).Commercial hypotensive agents, Xalatan® (latanoprost, 0.005%),Trusopt® (dorzolamide chlorhydrate, 2%), Timoftol® (timololmaleate, 0.5%) and Pilocarpine® (pilocarpine, 2%) were assayedby applying a volume of 40 μL. Since the tonometry method mayproduce discomfort in the rabbits, corneas were anaesthetized byapplying 10 μL of 1:10 (v:v) of oxibuprocaine/tetracaine (4 mgand 1 mg respectively, from ALCON-CUSI, Barcelona, Spain).

Experiments were performed following a blinded design: Novisible indication was given to the experimenter as to the appliedsolution (agent or vehicle). Intraocular pressure measurementswere made using a Tonopen® XL contact tonometer at baseline(pretreatment) and at the indicated times, following instillationof compound. Intraocular pressure was followed up to 6 h, inorder to study the time-course of the effect. On any given day,only a single dose was tested on a single animal, which waswashed out at least 2 days between doses.

2.4. Analysis of data

Numerical values are given as mean ± S.E.M. Group meanswere compared using Student's t-test (unpaired, two-tailed,unless otherwise stated) with a 5% fiducial point of signifi-cance, unless otherwise stated. The duration of the effect,termed mean-time effect, was calculated by measuring the timebetween 50% of intraocular pressure decrease after the drugtreatment and 50% of intraocular pressure recovery.

3. Results

3.1. Effect of PTD4-profilin on rabbit intraocular pressure

In order to study the effect of PTD4-profilin on rabbit'sintraocular pressure, a single dose of the compound was assayed

Table 1Comparative effects of PTD4-profilin to commercial anti-glaucoma drugs

Compound Maximal effect (mm Hg) Mean-time effect (h)

Control 22.1±0.6 –PTD4-Pfln 18.0±0.8a 7.1±0.7Xalatan® 17.1±1.3a 4.9±0.5Trusopt® 18.0±0.9a 4.0±0.5c

Timoftol® 14.3±1.2b 5.2±0.6Pilocarpine® 16.4±0.92a 4.2±0.5c

Values represent the mean±S.E.M. of six independent experiments. aPb0.05,bPb0.01, versus the control group. cPb0.05 versus PTD4-profilin.

147M. Morales et al. / European Journal of Pharmacology 567 (2007) 145–148

under the conditions described in methods. As it can be ob-served in Fig. 1, the application of PTD4-profilin produced asignificant reduction in intraocular pressure that was maximal2 h after instillation of the compound. The effect of PTD4-profilin produced a 20% reduction on intraocular pressurecompared to vehicle, remaining significantly below the controlvalue during at least the following 7.1 h (n = 6).

As a control, the same experiment was performed, assayingindependently only the transduction domain fused to a hema-toglutinin domain (PTD4-HA) or profilin without the PTD4domain. In any condition, intraocular pressure did not show signi-ficant changes compared to the vehicle (n = 4, Fig. 1). These resultstogether with the ones obtained for PTD4-profilin, demonstratethat only profilin fused to a transduction domainwas able to reduceintraocular pressure in New Zealand white rabbits.

3.2. Comparative study of PTD4-profilin effect with commercialavailable compounds

Since PTD4-profilin was able to reduce intraocular pressurein rabbits, we compared the effect of this new compound to otherdrugs commonly used for the treatment of ocular hypertension.Xalatan®, Trusopt®, Timoftol® and Pilocarpine®, four differentpharmaceutical products with different pharmacological me-chanisms, were tested in their ability to modify intraocularpressure (n = 6). As it can be observed in Table 1 in which themaximal effects of each of these substances are presented, all theassayed compounds reduced intraocular pressure significantly,being the best one Timoftol®. The reductions in intraocularpressure were between 35% (Timoftol®) and 18% (Trusopt®),compared to vehicle (saline).

When considering the interval of effect depicted by PTD4-profilin compared to the commercial compounds, the proteinpresented a mean-time effect of 6.8±1.1 h while Xalatan,Trusopt, Timoftol and Pilocarpine presented mean-time valuesof 4.9±0.5 h, 4.0±0.5 h, 5.2±0.6 and 4.2±0.5 h, respectively(n=6, Table 1).

The observation that the hypotensive effect of PTD4-profilinwas comparable to anti-glaucomatous commercial drugs in-creases the interest of this compound as a hypotensive agent forthe treatment of ocular hypertension associated to glaucoma.

4. Discussion

The present experimental work describes for the first time theeffect of the conjugate PTD4-profilin as an active protein able to

significantly reduce intraocular pressure in New Zealand whiterabbits. Also, the comparison of effects demonstrates that in ourexperimental model the effect of PTD4-profilin reduces intra-ocular pressure in a similar extent as commercial drugs do interms of maximal effect. The mean-time effect of PTD4-profilinwas remarkably longer when compared to the other compoundstested. It seems that PTD4-profilin kept a more sustainedreduction of intraocular pressure probably because it is using adifferent molecular mechanism to reduce this parameter. In clearcontrast with the mechanisms used by commercial compoundsavailable, we suggest a mechanism of action for PTD4-profilinon the cytoskeleton of trabecular meshwork cells, supposedlymodifying cell shape or contractility and therefore, reducing thehydrodynamic resistance to the aqueous humour outflow. In aprevious report we have demonstrated that PTD4-profilin per-fused in bovine anterior segments increased outflow facilityby modifying the trabecular meshwork permeability (Gomez-Cabrero et al., 2005). The observed effect on intraocularpressure suggests that the most likely target of PTD4-profilinafter a topical corneal application would be the trabecularmeshwork, although we cannot discard possible effects overother ocular structures related with the aqueous humour pro-duction or drainage.

PTD4-profilin reduces intraocular pressure about 20% asdemonstrated in results. It is noteworthy the lack of effect inintraocular pressurewhen the transduction domain (PTD4-HA) orthe recombinant protein lacking the transduction domain (pro-filin) was assayed. It is conceivable that PTD4 can pass throughthe different corneal layers, but the peptide itself does not haveany measurable effect of intraocular pressure. In contrast, the lackof effect of profilin alone is probably due to its impossibility tocross biological membranes or the corneal structure.

It is especially interesting to consider the biochemical andpharmacological properties of the tandem PTD4+profilin. Thecombination of these two molecules provides a fascinating newconcept, in clear contrast to the available drugs for the treatmentof ocular hypertension. From the chemical point of view, theseare proteins and not organic compounds. The protein profilin isnot acting either stimulating a receptor (like carbachol), anta-gonising it (like timolol) or inhibiting an enzyme (like dorzo-lamide) (Husband and Worsley, 2005). In fact, profilin is likelymodulating the cytoskeletal architecture and therefore, theoutflow tissues properties to enhance aqueous humour drainage.This mechanism of action is similar to other anti-glaucomatousdrugs under current development, such as latrunculins (Petersonet al., 2000b).

Other relevant aspect is the nature of the carrier proteinPTD4. This protein provides an extraordinary mechanism forprotein or peptide delivery inside the eye. The diffusion of smallmolecules through the cornea to reach the anterior and posteriorchamber is relying on structural properties such as size (MW),charge and solubility. The exciting properties of the PTD4peptide, allowing the transport of proteins such as profilin, openthe possibility of using it as a new ocular delivery system forthose molecules that present difficulties to pass into the eye. Theuse of this transduction technique for protein therapy has beenexplored for several different pathological conditions such as a

148 M. Morales et al. / European Journal of Pharmacology 567 (2007) 145–148

renal carcinomas or apoptotic ischemia. Moreover, the nature ofthe carried cargos varies from small peptides to large proteins oreven antibodies or liposomes (Zhao and Weissleder, 2004).

It has been previously reported that epithelial corneal cellscan act as a barrier to penetration of transduction proteins whenTAT-β-galactosidase was used (Guo et al., 2004), and that thisbarrier can be overcome by disrupting the corneal epithelium. Incontrast, our results suggest that PTD4-Pfn was able to cross thecorneal epithelium and the rest of cell layers to reach the eyeanterior chamber. Several reasons can account for these dif-ferences. First, we have employed a mutated version of theoriginal transduction domain (TAT) with greater transductionefficiency (Ho et al., 2001). Second, the lower molecular weightof profilin compared with β-galactosidase (120 kDa) can alsoaccount for the differences observed.

In summary, we have described the effect of PTD4-profilinreducing intraocular pressure in New Zealand white rabbits. Thenature of the delivery method by means of a carrier peptide(PTD4) and the active molecule profilin, likely modifying cyto-skeletal dynamics, open a new perspective in the development ofnew compounds for the treatment of ocular hypertension andglaucoma.

Acknowledgements

This work has been supported by research grants from theMinisterio de Educación and Fondo de Investigaciones Sani-tarias (Spain): SAF2004-06119-C02-01 SAFV-2002-PN03517-O; BFI 2003-01190; FIS/ 031495; BFU2006-04169/BFI;BFU2006-15047 and BM05-102-0 from F. La Caixa, ConsorcioNanofarma and S-SAL-0305-2006 from Comunidad de Madrid.

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