Bandello F, Battaglia Parodi M (eds): Anti-VEGF.
`Dev Ophthalmol. Basel, Karger, 2010, vol 46, pp 39–53
`
`Antivascular Endothelial Growth Factor in
`Diabetic Retinopathy
`Pierluigi Iaconoa ⭈ Maurizio Battaglia Parodib ⭈ Francesco Bandellob
`aFondazione G.B. Bietti per l’Oftalmologia, IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico),
`Rome, and bDepartment of Ophthalmology, University Vita-Salute, Scientific Institute San Raffaele,
`Milan, Italy
`
`Abstract
`Diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) represent the most com-
`mon causes of vision loss in patients affected by diabetes mellitus. Diabetic retinopathy (DR) needs
`special attention because of its high public health impact and impact on quality of life of patients.
`Actually, laser retinal photocoagulation is the standard of care for the treatment of DR. However,
`laser treatment reduces the risk of moderate visual loss by approximately 50%, without a remark-
`able vision recovery. Thus, new approaches in the treatment of DR have been taken into account
`and, more specifically, the therapy employing antivascular endothelial growth factor (anti-VEGF)
`drugs could play a meaningful role. VEGF is a pluripotent growth factor that functions as an endothe-
`lial cell-specific mitogen and vasopermeability factor. Through these mechanisms VEGF plays a criti-
`cal role in promoting angiogenesis and vascular leakage. A high level of VEGF has been detected in
`eyes presenting DME and PDR, and thereby VEGF is an attractive candidate as therapeutic target of
`pharmacological treatment in the management of DR. In the current chapter, the concepts and
`results of anti-VEGF therapy in the treatment of the DME and PDR are presented.
`Copyright © 2010 S. Karger AG, Basel
`
`Diabetic retinopathy (DR) is considered the most frequent vascular disorder being
`detectable in about 40% of diabetic patients 40 years and older [1]. Today, DR
`is the leading cause of acquired blindness among young adults throughout the
`developed countries [2]. Population-based epidemiological studies have estimated
`that after 20 years, DR is recognized to a certain extent, and that after 30 years
`a proliferative DR is present in the 70% of patients with diabetes mellitus type 1
`[3]. The World Health Organization estimates that about 171 million persons are
`affected by diabetes with an expected doubling of prevalence expected in the next
`20 years [4].
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`Role of the Vascular Endothelial Growth Factor in Diabetic Retinopathy
`
`Hyperglycemia is the main factor involved in the pathogenesis of DR. It results in the
`production of glycation end products, activation of the polyol pathway, and altered
`transduction of cellular signals [5–7]. The following damage to endothelial cells and
`pericytes, through activation of oxidative and inflammatory mechanisms, produces
`diabetic microangiopathy affecting small-caliber retinal vessel [8]. These altera-
`tions result in the deregulation of the mechanism of flow control with subsequently
`hypoxia and accumulation of fluid in the retinal tissue. Hypoxia represents the likely
`major inducer of vascular endothelial growth factor (VEGF) gene transcription, but
`the overexpression of VEGF is also upregulated in response to high glucose, pro-
`tein kinase C activation, and glycation end products, all elements characterizing the
`impairment of glycometabolic control [5–7, 9].
`VEGF is a pluripotent growth factor that functions as an endothelial cell-specific
`mitogen and vasopermeability factor and through these mechanisms the VEGF plays
`a critical role in promoting angiogenesis and vascular leakage [10–13]. In DR, the
`impairment of the blood retinal barrier and the increased permeability are respon-
`sible for the diabetic macular edema (DME) and several investigations underline the
`active role of VEGF. By disrupting the intercellular tight junctions between the retinal
`endothelial cells, VEGF increases the extracellular accumulation of fluid from the
`intravascular compartment [8]. Moreover, VEGF shows a role in mediating active
`intraocular neovascularization.
`Elevations of VEGF levels in ocular fluids from human patients with tissue hypoxia
`and active neovascularization secondary to DR have been well documented [14]. The
`increased levels of VEGF decline when treatment with panretinal photocoagulation
`induces regression of neovascularization. Thus, these studies demonstrated a tempo-
`ral correlation between VEGF elevations and active proliferative retinopathy evidenc-
`ing the role of VEGF as a key mediator of intraocular neovascularization secondary
`to DR. In essence, VEGF is an attractive candidate as therapeutic target of pharmaco-
`logical treatment in the management of DR.
`
`Anti-VEGF Therapy in the Treatment of Diabetic Macular Edema and Proliferative
`Diabetic Retinopathy
`
`The VEGF molecular family includes five members: placental growth factor, VEGF-A,
`VEGF-B, VEGF-C and VEGF-D [15]. Each of the different factors may link one or
`more of three VEGF receptors. Moreover, between the different factors, VEGF-A plays
`a major role in angiogenesis and vascular permeability. Alternative splicing of VEGF
`gene produces nine VEGF-A isoforms (VEGF121, VEGF145, VEGF148, VEGF162, VEGF165,
`VEGF165b, VEGF183, VEGF189, VEGF206) and among them VEGF165 is the most abun-
`dantly expressed isoform and is detected as being mainly responsible for DR.
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`The pathway enclosed between VEGF gene transcription and the activation of the
`VEGF receptor is the object of a new therapeutic approach based on the use of the
`VEGF antagonist. Pegaptanib, ranibizumab, bevacizumab and VEGF Trap are mol-
`ecules that are able to directly bind the VEGF protein. A new and interesting thera-
`peutic approach is the employment of bevasiranib. This molecule, interfering with
`messenger RNA, interrupts the synthesis of the VEGF protein. Last of all, rapamycin,
`employed commonly as an immunosuppressive, anti-inflammatory or antimycotic
`drug, reduces the activity of VEGF molecules interfering with the promoting signal,
`the active synthesis of VEGF, and reduces the response of endothelial cells to VEGF.
`
`Ranibizumab
`
`Ranibizumab is an antigen-binding fragment (Fab) derived from a humanized anti-
`VEGF antibody and this Fab inhibits all biologically active isoforms and active prote-
`olytic fragments of VEGF-A. Currently, ranibizumab is approved by the Food and Drug
`Administration for the treatment of neovascular age-related macular degeneration.
`Chun et al. [16] reported the first pilot study exploring the effects of two dos-
`ing regimens of ranibizumab in eyes affected by clinically significant DME. Of 10
`patients enrolled, 5 received 0.3 mg ranibizumab and 5 received 0.5 mg ranibizumab
`at baseline and at 1 and 2 months. At month 3, 40% of patients gained more than 15
`letters, 50% gained more than 10 letters, and 80% obtained an improvement of at
`least 1 letter in best corrected visual acuity (BCVA). At month 3, the mean decrease
`in central retinal thickness was 45.3 and 197.8 μm in the low- and high-dose groups,
`respectively. Intravitreal injections of ranibizumab were generally well tolerated and
`no systemic adverse events were reported.
`Nguyen et al. [17] investigated the role of ranibizumab in DME in the open-label
`study READ-1 (Ranibizumab for Edema of the Macula in Diabetes: Phase 1). Ten
`patients with chronic DME received intraocular injections of 0.5 mg ranibizumab
`at baseline and at 1, 2, 4, and 6 months. The main outcome measures were changes
`in BCVA, central retinal thickness as assessed by optical coherence tomography
`(OCT) measurement at the 7-month examination. Mean and median values of BCVA
`improved at 7 months by 12.3 and 11 letters respectively. Compared to the baseline,
`mean foveal thickness showed a meaningful reduction decreasing from 503 to 257
`μm with a 85% reduction of the excess foveal thickness present at baseline. The injec-
`tions were well tolerated with no ocular or systemic adverse events.
`More recently, the results of the READ-2 study were reported. READ-2 was a pro-
`spective, randomized, interventional, multicenter clinical trial designed to compare
`ranibizumab with focal/grid laser, alone or in combination, in DME [18]. 126 patients
`were randomized to receive 0.5 mg ranibizumab, focal/grid laser photocoagulation
`or a combination of 0.5 mg ranibizumab and focal/grid laser. Group 1, 42 patients,
`received 0.5 mg ranibizumab at baseline and months 1, 3, and 5. Group 2, 42 patients,
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`received focal/grid laser photocoagulation at baseline and month 3 if needed (center
`subfield thickness was >250 μm). Group 3, 42 patients, received a combination of 0.5
`mg ranibizumab and focal/grid laser at baseline and month 3. The primary outcome
`was the change in BCVA at month 6 in comparison with baseline. At month 6, the
`group receiving ranibizumab alone showed a significant improvement in mean BCVA
`with respect to the patients receiving focal/grid laser. The group receiving combined
`therapy was not statistically different from groups 1 or 2. Resolutions of 50, 33, and
`45% of excess foveal thickening were assessed in groups 1, 2, and 3, respectively.
`The RESOLVE study was specifically designed to evaluate the efficacy and safety of
`ranibizumab 0.5 mg in patients with visual impairment due to DME. The RESOLVE
`trial (a randomized, double-masked, multicenter, phase 2 study assessing the safety
`and efficacy of two concentrations of ranibizumab compared with non-treatment
`control for the treatment of DME with center involvement) evaluated the effect of
`ranibizumab on retinal edema and visual acuity (VA) in 151 patients with clinically
`significant DME. Patients with a central macular thickness of ≥300 μm were random-
`ized to receive three monthly injections with either 0.3 or 0.5 mg ranibizumab or pla-
`cebo. After the three monthly intravitreal injections, treatment was administrated on
`pro re nata basis for 9 months. The primary endpoint in the 1-year study was visual
`function at 6 months. The study design allowed the investigator to double the dose
`of ranibizumab if after 1 month the resolution of macular edema was incomplete.
`Moreover, retinal photocoagulation could be administered if needed.
`The preliminary results were partially presented at the 2009 ARVO Meeting [19].
`During the 12-month follow-up period, mean BCVA increased and mean CRT
`decreased continuously over time. The mean change in BCVA from baseline to the
`12-month examination was –1.4 letters in the sham group. The groups receiving 0.3
`and 0.5 mg gained, respectively, 11.8 and 8.8 letters. In order to provide further clarity
`on the effectiveness of treatments based on administration of steroidal or anti-VEGF
`drugs in comparison to conventional laser treatment, the DRCR net has designed
`a randomized, multicenter clinical trial which addressed the effects on visual acu-
`ity and on central retinal thickness in four groups receiving, respectively, intravitreal
`ranibizumab alone or associated with laser photocoagulation or triamcinolone asso-
`ciated with the laser treatment or laser treatment alone.
`The study recruited 691 patients and examined a total of 854 eyes in a follow-up
`period of 2 years. Two hundred ninety-three eyes were randomized to received laser
`alone, 187 eyes were assigned to the group receiving 0.5 mg ranibizumab + prompt laser,
`188 eyes received 0.5 mg ranibizumab + deferred laser (at least 24 weeks), and 186 eye
`were included in the group receiving 4 mg intravitreal triamcinolone + prompt laser.
`At 1-year examination, the mean change in the visual acuity letter score respect
`to the baseline value showed a statistically significant improvement in the ranibi-
`zumab + prompt laser group (+9±11 letters) and ranibizumab + deferred laser group
`(+9±12) but not in the triamcinolone + prompt laser group (+4±13) compared with
`the laser group (+3±13).
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`Over the 2 years of follow-up a different correlation between visual acuity change
`and retinal thickness was observed in each group. A progressive reduction in mean
`central subfield thickness was noted in the laser group during the 24 months of fol-
`low-up; however, the mean change in visual acuity did not continue to increase from
`the 1- to 2-year visit as noted instead during the first year of follow-up.
`In the triamcinolone + laser group, during the first year of follow-up an improve-
`ment of visual function was associated with a significant reduction in CST whereas
`from the 1- to 2-year examination the mean CST increased in parallel with a visual
`acuity reduction.
`Ranibizumab groups showed a parallel visual acuity improvement associated
`with a CST reduction from baseline to 12-month visit and following the OCT results
`remained relatively stable up to 24-month examination and paralleled the visual acu-
`ity outcomes during this time.
`Intraocular hypertension and cataract surgery were more frequently noted in
`the triamcinolone + prompt laser group in comparison to groups receiving ranibi-
`zumab + laser or laser alone.
`The current large prospective randomized clinical trial confirms the preliminary
`promising results in the treatment of DME and suggests as a combined therapy might
`offer a more efficacious approach in this disorder where the multi-factorial pathogen-
`esis involves several processes [20].
`Actually, several multicenter international clinical trials (e.g. RESTORE, RIDE and
`RISE) are ongoing in order to evaluate the efficacy and safety of ranibizumab 0.5 mg
`as monotherapy or in combination with laser photocoagulation in eyes affected by
`DME.
`With regard to the effects of ranibizumab on proliferative diabetic retinopathy
`(PDR), no studies are available in the literature evaluating this topic. However, DRCR.
`net has designed a prospective, randomized, comparative clinical trial to evaluate the
`role of ranibizumab or triamcinolone intravitreal injection as adjunctive treatment to
`panretinal photocoagulation for PDR.
`
`Pegaptanib
`
`Pegaptanib is a pegylated 28-nucleotide RNA aptamer that binds to the VEGF164/165
`isoform at high affinity. VEGF165 levels are present in human eyes affected by DR
`with increased concentration and play an active role in promoting angiogenesis and
`in enhancing vascular permeability. Initially, pegaptanib was employed only in the
`treatment of neovascular age macular degeneration, where it obtained the approval of
`the Food and Drug Administration. Considering the role of VEGF165 in DR and the
`safety and tolerability profile of intravitreally administered pegaptanib, a phase II trial
`was specifically designed to investigate the effects of pegaptanib in the management
`of DME.
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`The Macugen Diabetic Retinopathy Study was a randomized, sham-controlled, dou-
`ble-masked, dose-finding phase II trial designed to evaluate the effect of three doses of
`intravitreal pegaptanib vs. sham injection in patients affected by clinically significant
`DME [21]. The patients were randomized to receive 0.3, 1.0 or 3.0 mg of pegaptanib or
`sham injection at baseline, week 6 or week 12. If needed, further injections were admin-
`istrated every 6 weeks up to a maximum of three additional injections. Retinal laser
`photocoagulation could be delivered if the investigators judged it to be necessary. The
`main outcome measures were changes in BCVA, central retinal thickness as assessed
`by OCT measurement, and additional therapy with photocoagulation between weeks
`12 and 36. At the final visit at week 36, the group of patients receiving pegaptanib 0.3
`mg was significantly superior to sham injection, as measured by mean change in VA
`(+4.7 vs. –0.4 letters; p = 0.04), proportions of patients gaining >10 letters of VA (34 vs.
`10%; p = 0.003), change in mean central retinal thickness (68 μm reduction vs. 3.7 μm
`increase; p = 0.02). Moreover, only 25% of patients receiving pegaptanib required reti-
`nal photocoagulation in comparison with 40% of patients receiving a sham injection (p
`= 0.04). It is noteworthy that patients receiving 1.0 or 3.0 mg did not show a significant
`improvement compared to 0.3 mg with regard to BCVA or CRT changes. In general,
`pegaptanib was well tolerated at various concentrations; endophthalmitis occurred in 1
`of 652 injections and was successfully treated without severe visual loss.
`The Macugen Diabetic Retinopathy Study also provided new information on the
`ability of pegaptanib sodium to lead to regression of retinal neovascularization in
`proliferative DR [22]. Of 16 patients having retinal neovascularization in the study
`eye at baseline, 13 were assigned to receive pegaptanib treatment and 3 were assigned
`to sham injections. At 36 weeks, 8 of 13 (62%) in the pegaptanib treatment group
`showed regression of neovascularization, as assessed by fundus photography or fluo-
`rescein angiography, whereas no such regression occurred in 3 sham-treated eyes.
`In 3 of 8 with regression, neovascularization progressed at week 52 after cessation
`of pegaptanib at week 30, suggesting the necessity of repeated injections to control
`retinal neovascularization.
`More recently, Gonzalez et al. [23] reported the results of a prospective, random-
`ized, controlled, open-label, exploratory study designed to compare the efficacy of
`intravitreal pegaptanib vs. panretinal laser photocoagulation (PRP) in the treatment
`of active PDR. 20 subjects with active PDR were assigned at a 1:1 ratio to receive
`pegaptanib treatment in 1 eye every 6 weeks for 30 weeks or with PRP. In 90% of eyes
`randomized to pegaptanib, retinal neovascularization showed a complete regression
`by week 3. By week 12, in all eyes receiving pegaptanib a complete regression of retinal
`proliferation was obtained and preserved through week 36. In the PRP-treated group,
`at the 9-month examination, 25% demonstrated complete regression, 25% showed
`partial regression, and 50% showed persistent active PDR.
`With regard to BCVA and although the difference between the groups was not
`statistically significant (p = 0.22), pegaptanib-treated eyes showed an increase of 5.8
`letters in BCVA at 36 weeks, whereas the PRP-treated eyes lost 6.0 letters.
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`Bevacizumab
`
`Bevacizumab is a full-length recombinant humanized antibody active against all iso-
`forms of VEGF and currently it is approved for the treatment of metastatic colon
`cancer. However, anti-VEGF treatment approved for wet AMD, ranibizumab and
`pegaptanib is restricted in many countries; this situation has induced many retinal
`specialists to off-label use of bevacizumab and contemporarily it has allowed to under-
`line the role of this VEGF antagonist in many retinal disorders including neovascular
`AMD, macular edema in non-ischemic central retinal vein occlusion, pseudophakic
`cystoid macular edema and DR.
`Short-term effects of bevacizumab for DME in a large randomized phase II clinical
`trial were initially reported by the Diabetic Retinopathy Clinical Research Network
`[24]. 109 subjects with DME and Snellen acuity equivalent ranging from 20/32 to
`20/320 were prospectively enrolled and randomized to 1 of 5 groups: (a) focal photo-
`coagulation at baseline, (b) intravitreal injection of 1.25 mg bevacizumab at baseline
`and 6 weeks, (c) intravitreal injection of 2.5 mg bevacizumab at baseline and 6 weeks,
`(d) intravitreal injection of 1.25 mg bevacizumab at baseline and sham injection at 6
`weeks, or (e) intravitreal injection of 1.25 mg bevacizumab at baseline and 6 weeks
`with photocoagulation at 3 weeks.
`BCVA in groups receiving bevacizumab alone showed a median one-line improve-
`ment at the 3-week visit, which was preserved up to 12 weeks and was greater than
`the change in the group receiving only focal photocoagulation at baseline. A similar
`trend was observed with regard to central retinal thickness; comparing focal photo-
`coagulation vs. bevacizumab alone, a greater reduction in central retina thickness was
`observed in the bevacizumab groups at 3 weeks. Subsequently, only a trend towards a
`greater reduction at 6, 9, and 12 weeks was detected.
`No significant differences among groups receiving bevacizumab 1.25 vs. 2.5 mg for
`changes in BCVA or central retinal thickness were observed. Moreover, no meaningful
`differences were found comparing bevacizumab groups with groups receiving com-
`bined treatment in reduction in central subfield thickening or improvement in VA.
`Also, Lam et al. [25] evaluated the efficacy of two dosing regimens of bevacizumab at
`the 6-month follow-up. 48 patients were randomized to receive three monthly intra-
`vitreal injections of 1.25 or 2.5 mg bevacizumab. At each monthly scheduled visit
`a significant mean central foveal thickness reduction was observed in both groups.
`Similarly, mean logMAR BCVA showed a statistically significant improvement from
`baseline to the final visit at 6 months (from 0.63 to 0.52 in the 1.25-mg group and from
`0.60 to 0.47 in the 2.5-mg group). No significant difference in BCVA was observed
`between the two groups. Moreover, the study confirmed the effects of bevacizumab
`when the intravitreal injection reached a plateau of action at 3 weeks with a subse-
`quent decline, and repeated injections were necessary to maintain the initial effect.
`More recently, Arevalo et al. [26] reported the results of a retrospective, multicenter,
`interventional, comparative case series with a long-term follow-up that extended to
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`24 months. The study evaluated 139 eyes that underwent an intravitreal injection of
`1.25 or 2.5 mg bevacizumab. The main outcome measures were central foveal thick-
`ness as measured by OCT and changes in BCVA. Additional injections were adminis-
`tered if recurrence of macular edema was detected on OCT associated with VA loss.
`At 1 month, both groups showed a statistically significant improvement in BCVA
`and subsequently the gain was preserved up to the 24-month examination. The 1.25-
`mg group improved from 20/150 to 20/107 at 1 month, and to 20/75 at 24 months. In
`the 2.5-mg group, BCVA improved from 20/168 to 20/118 at 1 month, and to 20/114
`at the final visit. OCT examination evidenced a good anatomical response in both
`groups. At 1 month, the mean central macular thickness measurements decreased
`significantly from 446 to 333 μm; during the subsequent period a similar trend was
`observed up to 24 months with a final mean value of 279.7 μm. Similar results were
`observed in the 2.5-mg group. Over the 24-month follow-up period, 807 injections
`were performed and the mean number of injections per eye was 5.8 (range 1–15) at a
`mean interval of 12.2 ± 10.4 weeks.
`Long-term efficacy of repeated injections of intravitreal 1.25 mg bevacizumab for
`the treatment of chronic diffuse DME was also reported by Kook et al. [27]. The study
`included 126 patients affected by chronic, diffuse, clinically significant DME in part
`not responsive to previous treatments including laser photocoagulation (62% focal
`laser treatment, 38% panretinal laser treatment), triamcinolone intravitreal injection
`(41%) or vitrectomy (11%). 67 and 59 patients completed the scheduled visits to 6 and
`12 months, respectively. At the 6-month examination, logMAR BCVA ranged from
`a baseline value of 0.82 to 0.74 considering all patients. The mean BCVA of patients
`who completed the 12-month follow-up improved similarly from 0.82 to 0.74. Mean
`central retinal thickness decreased from 463 to 374 μm after 6 months, and to 357 μm
`after 12 months with a statistically significant difference. The authors concluded that
`even in cases with chronic diffuse ischemic DME not responding to other therapies,
`a successful treatment with repeated intravitreal injections of bevacizumab can be
`observed in a long-term follow-up.
`Other studies compared intravitreal bevacizumab treatment with intravitreal
`triamcinolone or focal retinal photocoagulation in refractory DME or as primary
`treatment. Paccola et al. [28] designed a randomized, prospective study in order to
`evaluate the anatomical response and VA outcomes after a single intravitreal injec-
`tion of 4 mg triamcinolone acetonide or 1.25 mg bevacizumab in the refractory dif-
`fuse DME. The study enrolled 26 patients; at baseline, logMAR BCVA was 0.936
`and 0.937 in the triamcinolone and bevacizumab groups, respectively. At 6 months,
`BCVA improved to 0.91 and 0.92 without achieving a significant difference; how-
`ever, interim analysis at the 1-, 2- and 3-month examinations evidenced a signifi-
`cant improvement in the triamcinolone group in comparison with the bevacizumab
`group. Central macular thickness was significantly reduced in the intravitreal triam-
`cinolone group compared with the bevacizumab group at weeks 4, 8, 12 and 24. The
`analysis of changes in CMT over the follow-up showed a significant from baseline
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`at weeks 4, 8 and 12 in the triamcinolone group and at weeks 4 and 8 in the bevaci-
`zumab group.
`A similar prospective and comparative case series was reported by Shimura et
`al. [29]. The study recruited 14 patients with bilateral long-standing DME; in each
`patient, 1 eye was selected to receive a single intravitreal injection of triamcinolone
`(4 mg) and the other to receive a single intravitreal bevacizumab injection (1.25 mg).
`logMAR BCVA in the triamcinolone group improved significantly from 0.64 to
`0.33 at 1 week, and the gain was subsequently preserved up to 12 weeks. At a final
`observation period of 24 weeks, BCVA decreased to 0.47 but was still significantly dif-
`ferent from the baseline value. Similarly, BCVA in the bevacizumab group improved
`from 0.61 to 0.39 at 1 week and maintained the initial gain up to 4 weeks. At 12 weeks,
`BCVA returned to the initial level. No further decrease or improvement was observed
`in the following 3 months. Between the two groups, a statistically significant differ-
`ence of BCVA was observed in favor of the triamcinolone group at 3 and 6 months.
`Morphological analysis revealed a significant decrease of foveal thickness from
`522 to 342.6 μm in the triamcinolone group at 1 week. At 12 weeks the foveal thick-
`ness maintained the improvement, but at 6 months it increased slightly to 410.4 μm.
`The bevacizumab group showed a significant foveal thickness reduction at 1 week
`from 527 to 397 μm and preserved the improvement also at 4 weeks. In the following
`weeks, the foveal thickness showed a progressive worsening and reached the initial
`level at 12 weeks; no further worsening or improvement was observed in the follow-
`ing 12 weeks. Between the two groups, a statistically significant difference of foveal
`thickness was observed in favor of the triamcinolone group at 1, 3 and 6 months.
`A more recently, randomized, three-arm clinical trial comparing intravitreal beva-
`cizumab injection (1.25 mg) alone or in combination with intravitreal triamcinolone
`acetonide (2 mg) versus macular laser photocoagulation as a primary treatment of DME
`was published by Soheilian et al. [30]. Each arm enrolled 50 patients receiving the treat-
`ment at baseline and at 12 weeks as needed. The main outcome measure was change in
`BCVA at the 4-month examination, but the study also provided the results at 36 weeks.
`The bevacizumab group showed a significant BCVA improvement from 0.71 to 0.54 at 6
`weeks; the initial gain was maintained at each following visit at 12, 24 and 36 weeks. The
`patients who underwent to combined treatment showed a significant BCVA improve-
`ment from 0.73 to 0.60 at 6 weeks. This group maintained BCVA also at 12 weeks but
`lost the statistically significant improvement at 6 and 9 months. In the macular photoco-
`agulation group, BCVA showed a stabilization at 6 weeks in comparison to the baseline
`value (0.60 vs. 0.55) and similar values were observed at all follow-up visits; however, we
`should consider that the three groups differed with regard to the baseline VA values.
`The mean values of central macular thickness decreased significantly in all groups
`only at 6 weeks in comparison to the baseline values and although the reduction was
`greater in the bevacizumab group with respect to the other two groups, no statisti-
`cally significant differences were registered at any of the follow-ups among the three
`groups. Retreatment was administered to 27 eyes up to 6 months and specifically 14
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`eyes received an additional bevacizumab injection, 10 eyes received combined treat-
`ment and macular photocoagulation was performed in 3 eyes.
`Another interesting element of this study is represented by regression of retinal
`neovascularization that presented initially in 9 patients. After the first bevacizumab
`treatment, the neovascularization resolved in all subjects. Treatment of retinal neo-
`vascularization associated with DR has been the object of study in several investiga-
`tions exploring the employment of bevacizumab as an antiangiogenic drug.
`In a retrospective study, Avery et al. [31] investigated the biologic effects of intra-
`vitreal bevacizumab in patients with retinal and iris neovascularization secondary to
`diabetes mellitus in 45 eyes. The patients received intravitreal bevacizumab in a dose-
`escalating regimen (6.2 g to 1.25 mg). Changes in fluorescein angiographic leakage
`and in BCVA were the primary and secondary outcome, respectively. At the 1-week
`examination, fluorescein angiography disclosed a complete or at least partial reduction
`in leakage of the neovascularization. Recurrence was registered in variable time: in 1
`case recurrence of retinal neovascularization was detected after 2 weeks whereas in
`the other cases no recurrent leakage was registered at the last follow-up of 11 weeks.
`In a prospective, non-randomized open-label study, Jorge et al. [32] evaluated the
`effects of bevacizumab in patients affected by active proliferative DR refractory to
`laser treatment and with a BCVA value inferior to 20/40. Each patient received a sin-
`gle intravitreal bevacizumab injection of 1.5 mg. 15 patients completed the scheduled
`visits up to 12 weeks of follow-up. Mean logMAR BCVA improved significantly from
`0.90 at baseline to 0.76 at week 1; subsequently the improvement was preserved up to
`the 12-week examination. At baseline the mean neovascularization leakage area was
`27.79 mm2. At the 1- and 12-week examinations, the mean neovascularization leak-
`age area decreased significantly to 5.43 and 5.50, respectively.
`Additional data on the use of bevacizumab in patients affected by proliferative
`DR complicated by vitreous hemorrhages has been provided by Moradian et al. [33].
`38 patients were enrolled and prospectively followed up to 20 weeks. Mean logMAR
`BCVA increased from 1.13 to 0.86 1 week after the injection; a further improvement
`was observed at 6, 12 and 20 weeks, with a final mean value of 0.53. Vitreous hemor-
`rhages resolved significantly at the 1- and 12-week examinations and about 50% of
`patients showed a complete resolution. At 20 weeks, only the 23% of the eyes pre-
`sented a slight degree of vitreous hemorrhages.
`A more recent investigation corroborates these results. Huang et al. [34] evaluated
`the efficacy of intravitreal bevacizumab combined with panretinal photocoagulation
`in the treatment of PDR complicated by vitreous hemorrhage. In their prospective
`study, 40 patients underwent an intravitreal bevacizumab injection (1.25 mg) fol-
`lowed by PRP. An additional injection was administered if no signs of decreased vit-
`reous hemorrhages were noted. In the event of persistent vitreous hemorrhage over
`12 weeks, vitrectomy was performed. The vitreous clear-up time and rate of vitrec-
`tomy were registered and compared with the control group treated with conventional
`methods. The vitreous clear-up time in the bevacizumab group was significantly
`
`48
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`Iacono · Battaglia Parodi · Bandello
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`Regeneron Exhibit 2012
`Page 10 of 15
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`lower than in the control group (11.9 vs. 18.1 weeks). Similarly, the bevacizumab
`group required vitrectomy in 10% of the patients compared to 45% of the control
`gro