Adv Ther (2015) 32:356–369
`DOI 10.1007/s12325-015-0205-5
`
`ORIGINAL RESEARCH
`
`A 1-Year Randomized Study of the Clinical
`and Confocal Effects of Tafluprost and Latanoprost
`in Newly Diagnosed Glaucoma Patients
`
`Paolo Fogagnolo . Angelica Dipinto . Elisa Vanzulli .
`Emanuele Maggiolo . Stefano De Cilla’ . Alessandro Autelitano .
`Luca Rossetti
`
`To view enhanced content go to www.advancesintherapy.com
`Received: February 23, 2015 / Published online: April 19, 2015
`Ó The Author(s) 2015. This article is published with open access at Springerlink.com
`
`ABSTRACT
`
`Introduction: The aim of the present study was
`to compare the confocal and clinical features of
`newly diagnosed glaucoma patients receiving
`unpreserved prostaglandins (tafluprost) versus
`preserved prostaglandins (latanoprost).
`Materials and Methods: 40 patients were
`randomized
`to
`tafluprost
`0.0015% (20
`patients;
`32
`eyes)
`or
`latanoprost
`0.005% ? benzalkonium chloride 0.02% (20
`patients; 35 eyes) once daily for 1 year.
`Inclusion
`criteria were
`new glaucoma
`diagnosis,
`and no ocular
`treatments
`for
`6 months before the study. Patients were
`evaluated at baseline and every 3 months with
`
`Trial registration: clinicaltrials.gov # NCT01433900.
`
`Electronic supplementary material The online
`version of this article (doi:10.1007/s12325-015-0205-5)
`contains supplementary material, which is available to
`authorized users.
`
`P. Fogagnolo (&) A. Dipinto E. Vanzulli
`E. Maggiolo S. De Cilla’ A. Autelitano L. Rossetti
`Eye Clinic, Dipartimento Testa-Collo, Ospedale San
`Paolo, University of Milan, Milan, Italy
`e-mail: fogagnolopaolo@googlemail.com
`
`S. De Cilla’
`Unit of Ophthalmology, Ospedale Maggiore della
`Carita`, Novara, Italy
`
`evaluation,
`ophthalmologic
`complete
`a
`Schirmer’s test, break-up time test, confocal
`microscopy
`of
`the
`central
`cornea,
`and
`measurement of
`intraocular pressure (IOP).
`Investigators were masked to treatment. Both
`eyes were analyzed if they fulfilled inclusion
`criteria. Treatments and changes between
`follow-up and baseline were compared by
`analysis of variance (ANOVA), t test and Chi-
`square test.
`Results: At baseline, the two groups had similar
`
`age, ocular
`
`surface and confocal findings;
`
`keratocyte activation was present
`
`in 40%,
`
`branching pattern in 85%, and beading in
`
`75%, with no inter-group differences. At
`
`follow-up, no significant clinical changes were
`
`IOP by
`from a drop of
`detected, apart
`3.6–4.2 mmHg in the two groups (p\0.001,
`with no difference between treatments). Despite
`
`inter-treatment
`
`ANOVA
`
`for
`
`confocal
`
`microscopy being negative,
`
`subtle changes
`
`were present. During follow-up,
`
`all
`
`eyes
`
`without nerve branching pattern at baseline
`
`progressively developed it when treated with
`
`latanoprost, whereas no change occurred using
`tafluprost treatment (p = 0.05). None of the eyes
`without beading at baseline developed it at the
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`Adv Ther (2015) 32:356–369
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`357
`
`the study in the tafluprost group,
`end of
`whereas beading did occur in 75% of patients
`(p = 0.05). Both
`treated with latanoprost
`treatments were associated with increased
`keratocyte activation at follow-up; the change
`from baseline was statistically significant after
`month 3 with latanoprost (p = 0.02) and after
`month 6 with tafluprost (p = 0.04).
`Conclusions: The two study treatments had
`similar clinical effects, but tafluprost had a
`more favorable profile for
`some confocal
`parameters of the cornea.
`Funding: Merck Sharp & Dohme International.
`
`Keywords: Confocal microscopy;
`Glaucoma;
`Intraocular
`pressure
`Latanoprost; Tafluprost
`
`Cornea;
`(IOP);
`
`INTRODUCTION
`
`intraocular pressure
`The beneficial effect of
`(IOP) lowering treatments to reduce glaucoma
`progression has been demonstrated by a
`number of multicenter,
`randomized studies
`[1–4]. On the other hand, more recent studies
`have also shown the detrimental effects of
`medical treatments for glaucoma on the ocular
`surface
`[5–11].
`It has been shown that
`prostaglandin analogs have
`inflammatory
`effects [5–9, 11], yet the vast majority of side
`effects are due to preservatives, in particular
`benzalkonium chloride (BAK), which is the most
`toxic and most used of ophthalmic preservatives
`[5–11]. BAK effects are dose dependent [7–12],
`and this is relevant considering that most
`glaucoma patients receive more than one IOP-
`lowering treatment [4]. Chronic BAK exposure is
`also associated with reduced efficacy of
`glaucoma surgery [13]. As a consequence,
`preservative-free treatments are preferable for
`glaucoma, as for all chronic eye diseases [14].
`
`Confocal microscopy is a recent technique
`which enables ophthalmologists
`to detect
`subtle inflammatory and toxic changes of the
`ocular
`surface [15]. By means of confocal
`microscopy, BAK has been shown to reduce
`the density of conjunctival goblet cells [16, 17],
`of conjunctival and corneal epithelial cells [17],
`and to deteriorate the normal characteristics of
`corneal nerves [18–20].
`Still, timing of occurrence of ocular surface
`changes when starting IOP-lowering treatments
`is an unexplored issue. Tafluprost is the most
`recent
`unpreserved
`prostaglandin
`analog
`introduced in clinical practice and it
`is
`characterized by the absence of BAK.
`To the best of the author’s knowledge, this is
`the first study to investigate and compare, from
`both clinical and confocal viewpoints,
`the
`effects
`of
`preserved
`and
`unpreserved
`prostaglandin analogs
`in newly diagnosed
`glaucoma patients with normal ocular surface.
`
`MATERIALS AND METHODS
`
`A randomized, masked, prospective study was
`carried out to test the primary hypothesis that
`treatment with
`preserved
`prostaglandins
`induces confocal changes of the cornea (both
`stromal inflammation and toxic damage to the
`sub-basal nerves) and that these anatomical
`changes would induce clinical changes, as
`detected
`during
`a
`general
`ophthalmic
`examination.
`
`Inclusion Criteria
`
`Inclusion criteria for the present study were:
`diagnosis of ocular hypertension (OH), primary
`open-angle
`glaucoma
`(POAG),
`pseudoexfoliative glaucoma or normal tension
`glaucoma, according to the definitions of the
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`Adv Ther (2015) 32:356–369
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`European Glaucoma Society Guidelines [21]; no
`previous treatments to reduce IOP and no
`treatment with any BAK-preserved eye drop for
`at
`least 6 months before
`the
`study; no
`fluorescein staining
`at baseline
`and no
`observable signs of ocular surface disease.
`
`Exclusion Criteria
`
`Exclusion criteria for the present study were:
`unwillingness
`to sign informed consent;
`aged\18 years; any ocular condition that was
`of safety concern or interfering with the study
`results; any ocular condition requiring the use
`of eye drops during follow-up (i.e., dry eye);
`closed/barely open anterior chamber angles or
`history of acute angle closure; ocular surgery or
`argon laser trabeculoplasty within the last year;
`ocular
`inflammation/infection
`occurring
`within 3 months prior
`to pre-trial
`visit;
`presence of the following ocular conditions:
`dry eye, moderate–severe blepharitis, Rosacea,
`Sjogren syndrome, pterygium or use of contact
`lens(es); hypersensitivity to BAK or to any
`other component of the trial drug solutions;
`any corneal pathology; diabetes at any stage;
`other
`abnormal
`condition
`or
`symptom
`preventing the patient from entering the trial
`(need
`for more
`than
`1
`IOP-lowering
`treatment), according to the investigator’s
`judgment; refractive surgery patients; women
`who were pregnant, of childbearing potential
`and not using adequate contraception or
`nursing; inability to adhere to treatment/visit
`plan.
`
`Clinical Plan
`
`visits
`5
`comprised
`protocol
`study
`The
`(performed at Eye Clinic of San Paolo Hospital,
`Milan, Italy): Baseline, Month 3, Month 6,
`Month 9 and Month 12.
`
`At baseline, a clinical evaluator performed a
`complete
`ophthalmologic
`evaluation
`to
`confirm diagnosis. The following examinations
`were done in the following sequence: anterior
`segment examination, Schirmer’s
`test and
`break-up time test. Thereafter, a confocal
`evaluator performed confocal microscopy of
`the
`central
`cornea.
`Finally,
`contact
`measurements were
`carried
`out
`in the
`following
`order:
`IOP,
`pachymetry
`and
`gonioscopy. A 15-min interval between two
`consecutive tests was observed.
`A study coordinator
`recorded medical
`history and then randomized patients into two
`groups: one group to receive unpreserved
`SaflutanÒ,
`(tafluprost
`0.0015%,
`Santen
`Pharmaceutical, Osaka, Japan) and one group
`to receive preserved prostaglandins (latanoprost
`0.005% ? BAK 0.02%, XalatanÒ, Pfizer S.r.L.,
`Latina,
`Italy) once daily
`to both eyes
`(randomization of 1:1, by means of a list of
`random numbers). Being patients treated to
`both eyes, a control group was not available.
`During the study, patients were instructed not
`to use any other topical treatment other than
`the study medication. The confocal and the
`clinical investigators were masked to treatment.
`Confocal and clinical examinations, as
`described above, were repeated at months 3, 6,
`9 and 12.
`Adherence to treatment, medical history,
`and side
`effects were
`checked by study
`coordinator at follow-up visits. Adverse effects
`were recorded. Symptoms were evaluated by
`means
`of
`comparison
`of
`ophthalmic
`medications
`for
`tolerability
`(COMTOL)
`questionnaire [22].
`
`Corneal Confocal Biomicroscopy
`
`The second version of Heidelberg Retina
`Tomograph
`(Heidelberg
`Engineering,
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`Adv Ther (2015) 32:356–369
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`359
`
`Heidelberg, Germany) is endowed with a lens
`system called the [Rostock Cornea Module
`(RCM)], and allows an in vivo confocal study
`of the ocular surface. The laser source used in
`the RCM is a diode laser with a wavelength of
`670 nm. The acquired two-dimensional images
`have a definition of 384 9 384 pixels over an
`area of 400 lm 9 400 lm with lateral digital
`resolution of 1 lm/pixel and a depth resolution
`of 2 lm/pixel.
`After administration of one drop of 0.4%
`oxybuprocaine and one drop of a lubricant gel
`(0.2% carbomer), the patient was asked to fixate
`on a small, bright, red light as the examination
`was performed in the contralateral eye. Correct
`alignment and contact with the cornea were
`monitored using the images captured by a
`camera tangential to the eye. The distance
`from the cornea to the microscope was kept
`stable using a single-use contact element in
`sterile
`packaging,
`(TomoCap, Heidelberg
`Engineering, Heidelberg, Germany).
`The
`examination took about 7 min per eye; 5
`images of each cornea layer and of the sub-
`basal layer were collected, both in central area.
`The highest resolution images taken of the
`different layers were considered for the analysis.
`Test–retest variability of confocal microscopy
`the central cornea was
`tested at
`the
`of
`beginning of the study using the following
`method. 5 eyes of 5 volunteers were tested 3
`times each: twice during the same day (at 9 a.m.
`and at 11 a.m.) and once the day after (at
`9 a.m.). The confocal operator evaluated these
`images and found an agreement of 80% or more
`for all parameters.
`
`Sample Size Calculation
`
`Given the paucity of information available on
`the
`effects of
`treatments with BAK-free
`prostaglandin on the ocular surface studied
`
`by confocal imaging, sample size calculation
`for this pilot study may be imprecise. The
`outcome
`of
`the
`study
`was
`corneal
`inflammation at confocal microscopy (defined
`as activation of anterior stroma, changes of
`nerve morphology, increase of dendritic cells).
`If a worth-detecting difference of 40% between
`the two groups is assumed, the presence of
`subclinical
`inflammation in 30% of normal
`cases, a one-tailed distribution in favor of the
`BAK-free arm of the study, a = 0.05, b = 0.2, a
`sample of 20 eyes would be necessary [20, 23,
`24]. It was decided to overpower the study
`including all treated eyes (a control group was
`absent in any case, being patients treated to
`both eyes), and this gave a study power of
`nearly 90%.
`
`Statistical Analysis
`
`All available data were analyzed (i.e., all eyes
`receiving study product were analyzed). The
`dataset was analyzed by means of linear and
`generalized, mixed-effect models of analysis of
`variance (ANOVA), with a post hoc test. In case
`of multiple comparisons, t test and Chi-square
`tests with Bonferroni–Holm correction were
`used. R open-access software was used (version
`3.1.3, R foundation for statistical computing,
`Vienna, Austria).
`
`Compliance with Ethics
`
`This present study was performed at the Eye
`Clinic, Department of Medicine, Surgery and
`Odontoiatry, San Paolo Hospital, University of
`Milan, Italy.
`All procedures followed were in accordance
`with the ethical standards of the responsible
`committee
`on
`human
`experimentation
`(University of Milan,
`Italy) and with the
`Helsinki Declaration of 1964, as revised in
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`2013. Informed consent was obtained from all
`patients for being included in the study.
`
`RESULTS
`
`Forty consecutive patients with new diagnosis
`of glaucoma or ocular hypertension were
`enrolled between January and July 2013. The
`study included 32 and 35 eyes in the tafluprost
`and
`latanoprost
`groups,
`respectively.
`Demographic
`characteristics of
`the
`study
`population and main study results are given in
`Tables 1, 2 and 3. The two groups had similar
`age and ocular surface and confocal findings at
`baseline (Figs. 1, 2). At the beginning of the
`study, activation of anterior stromal keratocytes
`was present in 40% of total patients (28% and
`50% of subjects in latanoprost and tafluprost
`p = 0.08);
`groups,
`respectively,
`branching
`pattern was present in about 85% of patients,
`and beading in 75% of cases.
`from treatment
`During a 1-year interval
`beginning, no significant clinical changes were
`detected, apart
`from a drop of
`IOP of
`3.6–4.2 mmHg in the two groups (p\0.001,
`
`Table 1 Demographic and main ocular features of the
`study population
`
`Tafluprost Latanoprost Total
`
`Number of
`patients
`
`Number of
`eyes
`
`20
`
`32
`
`20
`
`35
`
`40
`
`67
`
`Age years (SD) 68.5 ± 12.3 63.4 ± 14.4
`
`65.9 ± 13.5
`
`Sex f/m
`
`7/10
`
`8/10
`
`15/20
`
`Refraction
`
`0.98 ± 0.28 1.1 ± 0.28
`
`1.03 ± 0.28
`
`IOP mmHg
`(SD)
`
`18.5 ± 4.0
`
`18.5 ± 5.5
`
`18.5 ± 5.0
`
`IOP intraocular pressure, f/m female/male, SD standard
`deviation
`
`with no statistically significant difference
`between treatments; ANOVA).
`Confocal microscopy was similar between
`groups and between time points when analyzed
`by ANOVA. Yet, subtle changes occurring on
`the morphology of the cornea were shown at
`follow-up. All patients without branching
`pattern of
`sub-basal nerves
`at
`baseline
`progressively (from 9 to 12 months) developed
`this pattern when treated with latanoprost,
`whereas no change occurred at follow-up in
`(p = 0.04,
`subjects
`treated with tafluprost
`month 12). None of
`the patients without
`beading at baseline developed beading at the
`end of the study in tafluprost group, whereas
`this occurred in 6/8 (75%) patients treated with
`latanoprost (p = 0.05).
`Both treatments were associated with an
`increase of activation of anterior
`stromal
`keratocytes at
`follow-up;
`the change from
`baseline was statistically significant 3 months
`after
`starting
`treatment with latanoprost
`(p = 0.02)
`and
`6 months
`after
`tafluprost
`(p = 0.04).
`increase of
`A small and not significant
`dendritic cells density occurred over
`time,
`with no difference between treatments.
`No significant side effects were detected with
`any treatment during the study. No significant
`changes of symptoms were found, as evaluated
`by COMTOL scale, at follow-up in the two
`groups. Adherence to treatment was high
`(96%), and no study discontinuation occurred.
`
`DISCUSSION
`
`This paper explored the effects of tafluprost and
`latanoprost on a population of newly diagnosed
`POAG and OH with normal ocular surface, and
`the two treatments were found to have the same
`IOP-lowering effect and clinical tolerability over
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`Adv Ther (2015) 32:356–369
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`361
`
`1 year of follow-up, thus confirming previous
`findings [25–27].
`One novelty of the present study is that by
`means of a parallel randomization, prospective
`and masked design, the two treatments were
`also compared using confocal microscopy.
`Using this method,
`it was
`shown that a
`subgroup of otherwise normal
`subjects at
`baseline have
`subclinical
`corneal patterns
`(activation of anterior
`stromal keratocytes,
`nerve beading and branching). The number of
`cases with activation of keratocytes increased
`over time, thus confirming previous findings on
`the pro-inflammatory effect of prostaglandin
`analogs (regardless of BAK) [23]. Of the changes
`occurring during follow-up on sub-basal nerves,
`beading and branching were significantly lower
`in patients receiving tafluprost. Another paper
`recently
`compared
`the
`corneal
`confocal
`findings of the two treatments using a non-
`randomized design, and showed that tafluprost
`has a favorable safety profile [24].
`The main difference between the two study
`treatments is the absence of BAK in tafluprost
`formulation. BAK has been used for decades on
`nearly all ophthalmic formulations with an
`overall low percentage of serious side effects
`[28], even if recent studies demonstrated that
`BAK frequently causes relevant changes on the
`ocular surface, particularly when inspected by
`confocal microscopy [28].
`Little is known on the timing of occurrence
`of ocular surface changes when starting IOP-
`lowering treatments; in the present study it was
`shown that keratocyte activation (which was
`present at baseline in about one-third of eyes)
`increases immediately after the treatment is
`started and it tends to increase over time,
`whereas morphological changes of the nerves
`are present only after 9–12 months.
`found in
`Most of
`the corneal changes
`confocal studies on patients with glaucoma
`
`3/29
`
`1/34
`
`3/29
`
`5/30
`
`8/24
`
`0/35
`
`2/30
`
`5/30
`
`0/32
`
`2/33
`
`Punctatekeratitis,(yes/no)
`
`CIconfidenceinterval,SDstandarddeviation
`Inter-treatmentandintra-treatmentANOVA(mixed-effectmodels)notsignificant
`
`(13.3;15.4)
`
`(14.3;15.4)
`
`14.3±3.1
`(7.1;9.2)
`7.3±3.1
`
`14.9±1.7
`(8.7;11.2)
`
`7.0±3.7
`
`(14;15.6)
`14.8±2.3
`
`(14.2;15.4)
`
`14.8±1.8
`
`(7.1;9)
`7.1±2.7
`
`(7.1;9)
`7.1±2.9
`
`(13.2;15.1)
`
`14.2±2.7
`(6.8;8.7)
`7.8±2.9
`
`(13.1;20.7)
`16.9±10.9
`
`(14.6;21.4)
`
`18±10.1
`
`(14.2;21.4)
`17.8±10.3
`
`(14.9;20.7)
`
`17.8±8.8
`
`(10.6;17.9)
`14.3±10.4
`
`(13.4;15)
`14.2±2.4
`(6.6;8.3)
`7.5±2.5
`(15.1;21)
`18.1±8.9
`
`(7;8.8)
`7.6±2.6
`(12;19.4)
`15.7±10.7
`
`(12.8;14.5)
`
`13.6±2.5
`
`(13.9;15.4)
`
`14.7±2.2
`
`(6.2;8)
`7.1±2.8
`
`(16.9;20)
`18.5±4.5
`(6.6;8.8)
`7.7±3.1
`
`(16.7;20.3)
`
`18.5±5.4
`(6.3;9.1)
`7.7±4.2
`
`mean±SD(CI95%)
`
`IntraocularpressuremmHg
`
`(CI95%)
`
`Break-uptime,smean±SD
`
`LatanoprostTafluprost
`
`LatanoprostTafluprost
`
`LatanoprostTafluprost
`
`LatanoprostTafluprost
`
`Tafluprost
`
`Latanoprost
`
`Month12
`
`Month9
`
`Month6
`
`Month3
`
`Baseline
`
`Table2Clinicaldataofthestudypopulation
`
`(12.7;19.1)
`
`15.9±9.6
`
`(13.8;19.7)
`
`16.8±8.5
`
`(13.3;19.9)
`16.6±10.1
`
`mean±SD(CI95%)
`
`Schirmer’stest,mm
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`362
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`Adv Ther (2015) 32:356–369
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`27/5**
`
`27/5
`
`24/8*
`
`35/0
`
`28/7
`
`33/2
`
`27/5
`
`26/6
`
`24/8*
`
`32/3
`
`26/9
`
`33/2
`
`27/5
`
`26/6
`
`24/8
`
`0/3/7/10/12
`
`0/1/9/17/8
`
`1/5/6/14/6
`
`1/3/8/16/7
`
`0/1/9/15/7
`
`0/3/12/11/6
`
`0/3/9/16/7
`
`0/2/10/14/6
`
`0/2/6/17/10
`
`0/0/11/18/3
`
`0/4/9/16/6
`
`(2377;2756)
`
`2566±442
`
`(2263;2643)
`
`2453±307
`
`(2330;2623)
`
`2477±358
`
`(5.4;9.1)
`7.2±5.3
`(3.5;4.5)
`
`4±1.6
`
`(6.7;12)
`9.3±7.6
`(4.2;5.4)
`4.8±1.7
`
`(4.7;10)
`7.3±7.1
`(3.6;4.7)
`4.1±1.5
`
`(1980;2671)
`
`(2467;2796)
`
`(2157;2976)
`
`2325±610
`(5.9;10.6)
`
`8.3±7
`(3.8;5)
`4.4±1.7
`
`2632±325
`(5.5;11.9)
`
`8.7±8.9
`(4.1;5.4)
`4.8±1.8
`
`2566±724
`(5.9;12.4)
`
`(5728;6373)
`
`6051±886
`
`(5592;6103)
`
`5848±726
`
`(5766;6404)
`
`6085±892
`
`(5738;6200)
`
`5969±677
`
`(5942;6631)
`
`6286±947
`
`(5782;6219)
`
`6001±610
`
`Tafluprost
`
`Latanoprost
`
`Tafluprost
`
`Latanoprost
`
`Tafluprost
`
`Latanoprost
`
`Month12
`
`Month9
`
`Month6
`
`*p=0.05(inter-treatmentv2);**p=0.04(inter-treatmentv2)
`ANOVAanalysisofvariance,CIconfidenceinterval,SDstandarddeviation
`Inter-treatmentandintra-treatmentANOVA(mixed-effectmodels)notsignificant
`
`30/5
`
`27/5
`
`30/5
`
`27/5
`
`30/5
`
`Branchingpattern,(yes/no)
`
`24/11
`
`31/4
`
`21/11
`
`24/8
`
`21/14
`
`30/5
`
`16/16
`
`24/8
`
`(yes/no)
`anteriorstromalkeratocytes,
`
`10/25
`
`Presenceofactivationofthe
`
`27/8
`
`Sub-basalnervebeading,
`
`(yes/no)
`
`(grade0/1/2/3/4)
`
`0/2/12/10/11
`
`0/4/9/9/10
`
`0/6/11/7/11
`
`0/1/10/10/11
`
`1/1/11/17/5
`
`Sub-basalnervetortuosity,
`
`1/3/8/16/4
`
`0/1/9/19/6
`
`0/0/14/13/5
`
`2/2/8/20/3
`
`Sub-basalnervereflectivity,
`
`(grade0/1/2/3/4)
`
`(2469;2914)
`
`2692±520
`
`(2144;2764)
`
`2454±652
`
`(2503;2984)
`
`2743±588
`
`(2398;3577)
`2987±1276
`
`9.2±9.5
`(3.5;4.7)
`4.1±1.8
`
`(3.7;7.2)
`5.4±4.9
`(3.8;4.9)
`4.3±1.5
`
`(4.1;9.3)
`6.7±7.6
`(4.3;5.4)
`4.8±1.6
`
`(4;7.8)
`5.9±5.3
`(3.7;4.5)
`4.1±1.3
`
`(4.3;10.8)
`
`7.5±9.3
`(4.1;5.2)
`4.7±1.7
`
`(5902;6585)
`
`6244±939
`
`(5833;6326)
`
`6079±677
`
`(6026;6714)
`
`6370±928
`
`(5393;6207)
`5800±1136
`
`Tafluprost
`
`Latanoprost
`
`Tafluprost
`
`Latanoprost
`
`Month3
`
`Baseline
`
`mean±SD(CI95%)
`
`Endothelialdensitycells/mm2
`framemean±SD(CI95%)
`
`Densityofdendriticcellsper
`
`mean±SD(CI95%)
`Fiberdensityperframe
`mean±SD(CI95%)
`
`Epithelialdensitycells/mm2
`
`Table3Confocaldataofthestudypopulation
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`Adv Ther (2015) 32:356–369
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`363
`
`Fig. 1 Confocal images of a patient treated with tafluprost.
`a Sub-basal plexus at baseline. b Sub-basal plexus at month
`12: no relevant changes of density, length, morphology are
`
`shown. c Anterior
`stroma at baseline; no keratocyte
`activation is present. d Anterior stroma at month 12: no
`changes are shown; keratocyte activation is absent
`
`have been attributed to BAK. In particular, BAK
`has a dose-dependent apoptotic action [29]
`which has been shown to disrupt
`the
`epithelial barrier of both conjunctiva [16, 30]
`and cornea [11]; at ultrastructural levels, BAK
`induces a massive reduction of goblet cells [16,
`
`30] and an anatomical disruption of corneal
`glycocalyx and microvilli
`[11]. In the most
`severe cases, deeper layers of the ocular surface
`can also be
`involved by BAK exposure:
`conjunctival fibrosis and keratinization have
`been reported [31]. Most recently, BAK exposure
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`364
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`Adv Ther (2015) 32:356–369
`
`Fig. 2 Confocal
`treated with
`a patient
`images of
`latanoprost. a Sub-basal plexus at baseline. b Anterior
`stroma at baseline; no keratocyte activation is present.
`c Sub-basal plexus at month 12: disruption of normal nerve
`
`structure is shown: branching and beading are present, and
`nerve is tortuous; density is overall conserved. d Anterior
`stroma at month 12 showing keratocyte activation
`
`has been associated also with anterior chamber
`inflammation [32].
`From the literature, the use of BAK-free
`treatments is preferable in all cases [16, 18, 30,
`33, 34]. Studies comparing BAK and BAK-free
`
`treatments for glaucoma showed the superiority
`of BAK-free treatments on clinical findings [33,
`34] and, by means of confocal microscopy,
`conjunctival [16, 30] and corneal [18] findings.
`The non-randomized, cross-sectional paper by
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`Adv Ther (2015) 32:356–369
`
`365
`
`Martone et al. [18] was one of the first to suggest
`that patients receiving unpreserved treatments
`for glaucoma have confocal findings more
`similar to controls than to patients treated
`with BAK-preserved eye drops.
`Regardless of the exposure to BAK, it has
`been suggested that stromal activation may be
`facilitated by the pro-inflammatory activity of
`prostaglandin analogs [23]. Even if other studies
`found that activation may be similar for beta-
`blockers and prostaglandins [18, 20], the data
`seem to support the effect of the drug itself on
`the keratocyte activity.
`switching from
`The beneficial effect of
`prostaglandin
`preserved
`to
`unpreserved
`treatment has been explored by a recent study
`which showed, over a 1-year period, an increase
`in epithelial and nerve densities, a reduction of
`keratocyte activation, a reduction of bead-like
`formations and nerve tortuosity [25]. Despite
`these premises, the present study seems to
`indicate that
`such findings may not be
`clinically
`relevant
`for
`newly
`diagnosed
`glaucoma patients, without ocular
`surface
`disease,
`receiving low doses of BAK (i.e.,
`monotherapy)
`for a short period of
`time.
`Clinical
`data
`and
`symptoms,
`in
`fact,
`overlapped in the two study groups at all
`visits. The confocal difference of
`the two
`treatments may gain relevance in patients
`with longer
`follow-up, with concomitant
`ocular surface disease, or exposure to higher
`BAK concentrations due to concomitant use of
`preserved eye drops. These factors were outside
`the scope of the study, but these patients will
`have continued follow-ups to detect possible
`future clinical and confocal changes.
`Readers should be aware that this study
`reflects the limits of confocal microscopy, i.e.,
`subjectivity and limited repeatability. The area
`
`investigated by this device is also very small and
`may be not representative of the whole cornea.
`The data are comparable to those available in
`literature for corneal confocal microscopy of
`normal patients, with the exception of dendritic
`cells, which were lower in the present study
`sample than in literature (although Zhivov et al.
`[35]. suggested that dendritic cell density in
`normal subjects may range from 0 to 64/mm2).
`In general, data on confocal microscopy have a
`large span of normality, as shown in Table 4
`[35–45]. Moreover, the discrimination between
`normal and abnormal findings at confocal
`investigation is not
`always univocal;
`for
`example,
`the
`role played by branching,
`tortuosity or abnormally high or abnormally
`low reflectivity is debated [16, 18–20].
`Due to the paucity of data on confocal
`microscopy
`in
`newly
`treated
`glaucoma
`patients,
`sample
`size
`assumptions were
`approximate; the inclusion of all available eyes
`in analysis increased the statistical power of the
`study but
`could also limit
`its validity.
`Nevertheless, this paper has the merit of a
`randomized,
`double-blinded
`design;
`the
`confocal
`evaluators were blinded to the
`characteristics of the patients and evaluated
`images in a blinded fashion.
`
`CONCLUSION
`
`In conclusion, the present study found out that
`the low daily exposure to BAK of patients
`treated with latanoprost may facilitate the
`development of
`confocal
`changes of
`the
`cornea, which occurred less
`frequently on
`patients treated with tafluprost. Activation of
`anterior stromal keratocytes was present at
`baseline in one-third of cases and increased at
`follow-up,
`probably
`due
`to
`the
`pro-
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`
`Table 4 Values of normality for confocal findings in naı¨ve subjects
`Value of normality, mean – SD
`
`Parameter
`
`Basal epithelium (cells/mm2)
`
`Nerve fibers (fibers/frame)
`
`Nerve fiber density (fibers/mm2)
`
`Nerve tortuosity (grade 0–4)
`
`Nerve reflectivity (grade 0–4)
`
`Dendritic cell density (cells/mm2)
`
`Endothelial density (cells/mm2)
`
`SD standard deviation
`
`5623 ± 389
`
`5823 ± 602
`
`6333 ± 604
`
`8916.7 ± 645.8
`
`8996 ± 1532
`
`2.9 ± 0.8
`
`3.8 ± 0.7
`
`5.26 ± 1.3
`
`5.85 ± 2.04
`
`5.9 ± 0.7
`
`31.9 ± 94
`
`1.2 ± 0.39
`
`1.8 ± 0.7
`
`2.0 ± 0.8
`
`2.3 ± 0.6
`
`2.07 ± 0.9
`
`2.6 ± 0.9
`
`34 ± 3
`
`34.9 ± 5.7
`
`2539 ± 338
`
`2968 ± 385
`
`3105 ± 497
`
`References
`
`Martone et al. [18]
`
`Patel et al. [36]
`
`Ceresara et al. [37]
`
`Hu et al. [38]
`
`Eckard et al. [39]
`
`De Cilla` et al. [40]
`
`Bucher et al. [41]
`
`Martone et al. [18]
`
`Ceresara et al. [37]
`
`Kurbanyan et al. [42]
`
`Hertz et al. [43]
`
`Martone et al. [18]
`
`Kurbanyan et al. [42]
`
`De Cilla` et al. [40]
`
`Ceresara et al. [37]
`
`Martone et al. [18]
`
`De Cilla` et al. [40]
`
`Zhivov et al. [39]
`
`Lin et al. [44]
`
`Salvetat et al. [45]
`
`Ceresara et al. [37]
`
`Hu et al. [38]
`
`inflammatory activity of prostaglandin analogs.
`From a clinical viewpoint, the two treatments
`had similar IOP-lowering effect and tolerability.
`
`ACKNOWLEDGMENTS
`
`The paper was supported by the unrestricted
`grant # 00111760 by Merck Sharp & Dohme
`International. Article processing charges and
`the open access fee were supported by Santen
`
`Ltd. Registration number: NCT01433900 (at
`
`www.clinicaltrials.gov). All named authors
`
`meet the ICMJE criteria for authorship for this
`
`manuscript, take responsibility for the integrity
`
`of the work as a whole, and have given final
`
`approval to the version to be published. All
`
`authors had full access to all the data in this
`
`study and take complete responsibility for the
`
`integrity of the data and the accuracy of the
`data analysis. The Authors are grateful
`to
`
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`

`Adv Ther (2015) 32:356–369
`
`367
`
`Giovanni Montesano, MD, Universita` degli
`Studi di Milano, Milan, Italy for his help on
`statistical analysis of the results of the study.
`
`of
`
`Conflict
`
`interest. Paolo
`Fogagnolo
`received a speaker honorarium from Merck
`Sharp & Dohme International.
`Luca Rossetti received a speaker honorarium
`from Merck Sharp & Dohme International.
`Angelica Dipinto, Elisa Vanzulli, Emanuele
`Maggiolo, Stefano De Cilla’ and Alessandro
`Autelitano declare that they have no conflict
`of interest.
`
`Compliance with ethics
`
`guidelines. All
`procedures followed were in accordance with
`the
`ethical
`standards of
`the
`responsible
`committee on human experimentation (Ethics
`Committee of the University of Milan, Italy)
`and with the Helsinki Declaration of 1964, as
`revised in 2013. Informed consent was obtained
`from all patients for being included in the
`study.The Author(s)
`
`Open Access. This article is distributed
`under the terms of the Creative Commons
`Attribution Noncommercial License which
`permits any noncommercial use, distribution,
`and reproduction in any medium, provided the
`original author(s) and the source are credited.
`
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