© The American Society of Gene Therapy
`
`original article
`
`Ranibizumab for Macular Edema Due to
`Retinal Vein Occlusions: Implication of VEGF
`as a Critical Stimulator
`Peter A Campochiaro1, Gulnar Hafiz1, Syed Mahmood Shah1, Quan Dong Nguyen1, Howard Ying1,
`Diana V Do1, Edward Quinlan1, Ingrid Zimmer-Galler1, Julia A Haller1, Sharon D Solomon1,
`Jennifer U Sung1, Yasmin Hadi1, Kashif A Janjua1, Nida Jawed1, David F Choy2 and Joseph R Arron2
`
`1Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 2Immunology, Tissue Growth,
`and Repair Diagnostics and Biomarkers Genentech, Inc., South San Francisco, California, USA
`
`Macular edema is a major cause of vision loss in patients
`with central retinal vein occlusion (CRVO) or branch reti-
`nal vein occlusion (BRVO). It is not clear how much of
`the edema is due to hydrodynamic changes from the
`obstruction and how much is due to chemical media-
`tors. Patients with macular edema due to CRVO (n = 20)
`or BRVO (n = 20) were randomized to receive three
`monthly injections of 0.3 or 0.5 mg of ranibizumab. At
`the primary endpoint, month 3, the median improve-
`ment in letters read at 4 m was 17 in the 0.3-mg group
`and 14 in the 0.5-mg group for CRVO, and 10 and 18,
`respectively for the BRVO group. Optical coherence
`tomography (OCT) showed that compared to injections
`of 0.3 mg, injections of 0.5 mg of ranibizumab tended to
`cause more rapid reductions of central retinal thickening
`that lasted longer between injections, but in 3 months,
`excess central retinal thickening which is a quantita-
`tive assessment of the macular edema, was reduced by
`~90% in all four treatment groups. There was no correla-
`tion between the amount of improvement and duration
`of disease or patient age at baseline, but there was some
`correlation between the aqueous vascular endothelial
`growth factor (VEGF) level at baseline and amount of
`improvement. These data indicate that excess produc-
`tion of VEGF in the retinas of patients with CRVO or
`BRVO is a major contributor to macular edema and sug-
`gest that additional studies investigating the efficacy of
`intraocular injections of ranibizumab are needed.
`Received 28 September 2007; accepted 7 January 2008; published online
`5 February 2008. doi:10.1038/mt.2008.10
`
`INTRODUCTION
`The pathogenesis of central retinal vein occlusions (CRVOs) is
`poorly understood. In the early stages, there are scattered hemorr-
`hages throughout the entire retina, cotton wool patches, a sign of
`retinal ischemia, and massive edema of the retina. Fluorescein
`angiography often shows delayed filling of retinal veins suggesting
`
`reduced blood flow, staining of the walls of retinal veins, and leakage
`of dye into the retina. It has been concluded that this picture most
`likely occurs from thrombosis within the central retinal vein caus-
`ing partial obstruction of blood flow from the eye, increased intra-
`luminal pressure in the retinal veins, and increased transudation of
`blood and plasma into the retina. Histopathology has confirmed
`the presence of a thrombus in the central retinal vein in several
`cases.1 The marked increase in interstitial fluid and protein increases
`interstitial pressure and is likely to be an impediment to capillary
`perfusion resulting in ischemia. The subacute stage varies among
`patients depending primarily upon the amount of retinal ischemia,
`and patients are classified as ischemic or nonischemic, although it is
`not an all or none dichotomy. In some patients ischemia increases
`over time and they are viewed as undergoing a transition from nonis-
`chemic to ischemic. Severe retinal ischemia can be complicated by
`retinal neovascularization, iris neovascularization, neovascular glau-
`coma, and a very poor visual outcome. Thus the amount of retinal
`ischemia is one of the major determinants of outcome.
`Those patients classified as nonischemic still have retinal isch-
`emia, as demonstrated by cotton wool patches and areas of capil-
`lary nonperfusion seen in fluorescein angiograms. These patients
`often enter a chronic stage in which they have severe macular
`edema that may last for many months and often years. Eventually
`the edema may resolve, presumably because there is resolution of
`the venous obstruction due to recanalization and/or formation of
`collateral vessels, but generally the visual outcome is poor due to
`damage to macular photoreceptors from the chronic edema, poor
`perfusion of perifoveal capillaries, or both.2
`Hypertension is a major risk factor for branch retinal vein
`occlusions (BRVOs).3 Chronic hypertension leads to thickening
`of the walls of retinal arterioles and since retinal arterioles and
`veins share a common adventitia at crossings, this may cause
` constriction of retinal veins that can lead to occlusions.4,5 The
`complications of BRVOs are similar to those for CRVOs but more
`limited in scope, because only part of the retina is drained by the
`involved branch vein. There is increased luminal pressure distal
`to the obstruction resulting in increased transudation of blood
`and plasma, increased interstitial fluid pressure, and reduced
`
`Correspondence: Peter A. Campochiaro, Maumenee 719, The Wilmer Eye Institute, The Johns Hopkins University School of Medicine,
`600 North Wolfe Street, Baltimore, Maryland 21287-9277, USA. E-mail: pcampo@jhmi.edu
`
`Molecular Therapy vol. 16 no. 4, 791–799 april 2008
`
`791
`
`Mylan Exhibit 1036
`Mylan v. Regeneron, IPR2021-00881
`Page 1
`
`Joining Petitioner: Apotex
`
`

`

`Ranibizumab for Macular Edema in Vein Occlusions
`
`© The American Society of Gene Therapy
`
`Table 1 Baseline characteristics
`
`CRVO
`
`BRVO
`
`
`
`0.3 mg
`
`0.5 mg
`
`0.3 mg
`
`0.5 mg
`
`63 ± 17
`69
`34–83
`
`9 ± 7
`7.4
`1–26
`
`68 ± 13
`70
`48–83
`
`16 ± 17
`13
`0.5–53
`
`69 ± 13
`68
`43–84
`
`65 ± 10
`65
`50–82
`
`5 ± 3
`5
`0.4–9
`
`3 ± 2
`3
`0.8–6
`
`capillary perfusion causing ischemia. Since the area of ischemic
`retina tends to be much less than that in patients with CRVO, iris
`neovascularization and neovascular glaucoma are rare, but retinal
`neovascularization adjacent to the ischemic retina can occur and
`can cause vitreous hemorrhages necessitating scatter photocoagu-
`lation to the ischemic retina. The most common cause of reduced
`vision is macular edema, but nonperfusion of perifoveal capillar-
`ies can also be another contributing factor. Grid laser photoco-
`agulation to the poorly perfused retina adjacent to the fovea can
`help to resolve macular edema and improve vision.6
`Thus, in both CRVO and BRVO, retinal ischemia occurs and
`serves as an exacerbating factor. The ischemic retina releases
`vascular endothelial growth factor (VEGF) which underlies
`neovascular complications, but also causes excessive vascular
`permeability.7,8 Hence, the release of VEGF is likely to contrib-
`ute to macular edema. In this study, we investigated the potential
`contribution of VEGF to macular edema in patients with CRVO
`and BRVO by testing the effects of intraocular injections of ranibi-
`zumab (Lucentis; Genentech, South San Francisco, CA), an Fab
`fragment that binds and neutralizes all isoforms of VEGF-A.9
`
`RESULTS
`Baseline characteristics of study patients
`The baseline characteristics of patients are shown in Table 1. For
`CRVO patients, the 0.5-mg group was somewhat older than the
`0.3-mg group and had a longer duration of disease.
`
`Effect of ranibizumab on central retinal
`(foveal) thickening
`Figure 1 shows optical coherence tomography (OCT) scans at all
`time points up to the primary endpoint for five randomly selected
`patients with CRVO randomized to each dose. Although the
`response varied somewhat among patients in each group, most
`patients showed substantial reduction in central retinal thickness
`over time. Within 7 days of the first injection, 8 out of 10 patients
`in the 0.3-mg group and 9 out of 10 in the 0.5-mg group had a
`substantial improvement in edema as assessed by OCT (Figure 2a
`and b). Four patients in the 0.3-mg group showed a worsening of
`the thickening between the time points of 1 week and 1 month
`but improved after the next two injections, while the other six
`showed persistent improvement 1 month after the first injection
`that improved further with subsequent injections (Figure 2a). All
`the patients in the 0.5-mg dose group showed the latter pattern
`of response (Figure 2b). Figure 3 shows the OCT scans of the
`five randomly selected patients with BRVO randomized to each
`group. They tended to show rapid and sustained responses after
`the first injection of 0.3 or 0.5 mg of ranibizumab with further
`improvement after subsequent injections. Measurements of cen-
`tral retinal thickening confirmed that this was the case for most
`patients, but two in the 0.3-mg group and one in the 0.5-mg group
`showed slight worsening between the time points of 1 week and
`1 month (Figure 4).
`In patients with CRVO, the median excess foveal thickness
`was 340 μm at baseline in the 0.3-mg group and 7 days after the
`first injection it improved to 124 μm, thus eliminating 64% of the
`edema (Figure 5a). In the 0.5-mg group, the median excess foveal
`thickness improved from 309 μm at baseline to 53 μm 1 week after
`
`Age (years)
` Mean ± SD
` Median
` Range
`Duration of disease (months)
` Mean ± SD
` Median
` Range
`Systemic disease
` Diabetes
` Hypertension
` Hyperlipidema
` Elevated homocysteine
`Ocular disease
` Glaucoma
` Other
`Prior treatment
`0
` Bevacizumab
`1
` Steroids
`1
` Laser
`Visual acuity (ETDRS letters read at 4 m)
` Mean ± SD
`16 ± 13
` Median
`18
`Excess foveal thickness (in μm)
`297 ± 126 252 ± 104 288 ± 101
` Mean ± SD
`346 ± 88
`309
`270
`294
` Median
`340
`Abbreviations: BRVO, branch retinal vein occlusion; CRVO, central retinal vein
`occlusion; ETDRS, Early Treatment Diabetic Retinopathy Study.
`
`3
`5
`4
`1
`
`1
`2
`
`3
`6
`7
`3
`
`3
`5
`
`0
`2
`3
`
`3
`9
`7
`3
`
`0
`5
`
`1
`2
`4
`
`3
`8
`3
`6
`
`1
`3
`
`2
`2
`3
`
`23 ± 15
`26
`
`26 ± 12
`29
`
`20 ± 14
`23
`
`the first injection, thus eliminating 83% of the edema (Figure 5b).
`At the primary endpoint of 3 months, excess foveal thickness was
`reduced to 25 μm (eliminating 93% of the edema) in the 0.3-mg
`group, and 35 μm (eliminating 89% of the edema), in the 0.5-mg
`group. Thus, in patients with CRVO, edema was reduced to a
`greater extent and the reduction was more sustained after a single
`injection of 0.5 mg of ranibizumab compared to 0.3 mg, but after
`three injections of either dose, most of the edema, i.e., ~90%, had
`been eliminated.
`In patients with macular edema due to BRVO, the median
`excess foveal thickness was 270 μm at baseline in the 0.3-mg group
`and 7 days after the first injection it improved to 48 μm, thus eli-
`minating 82% of the edema (Figure 5c). In the 0.5-mg group, the
`median excess foveal thickness was 294 μm at baseline and 7 days
`after the first injection it improved to 51 μm, eliminating 83% of
`the edema (Figure 5d). At the primary endpoint, excess foveal
`thickness was essentially eliminated in each group.
`The last injection was at the time point of month 2; by month
`4 in some patients and month 6 in most patients there was recur-
`rent edema in patients with CRVO and worsening of the edema
`
`792
`
`www.moleculartherapy.org vol. 16 no. 4 april 2008
`
`Mylan Exhibit 1036
`Mylan v. Regeneron, IPR2021-00881
`Page 2
`
`Joining Petitioner: Apotex
`
`

`

`© The American Society of Gene Therapy
`
`Ranibizumab for Macular Edema in Vein Occlusions
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`Figure 1 Cross sections through the fovea obtained by optical coherence tomography in patients with central retinal vein occlusion. The hori-
`zontal cross sections at baseline (BL), day 7 (D7), month 1 (M1), month 2 (M2), and month 3 (M3, primary endpoint) are shown for five randomly
`selected patients of the 10 patients treated with (a) 0.3 mg or (b) 0.5 mg of ranibizumab.
`
`tended to occur sooner after the last injection in the 0.3-mg
`group compared to the 0.5-mg group. In the 0.3-mg/CRVO group,
`seven, and then five patients were given another treatment (usually
`an off-label injection of bevacizumab) at the time points of months
`4 and 6, respectively. In the 0.5-mg/CRVO group, three, and then
`six patients received additional injections at the time points of
`months 4 and 6, respectively. In patients with BRVO, recurrent
`edema was somewhat less frequent than in patients with CRVO,
`but still occurred in several patients. In the 0.3-mg/BRVO group,
`one, and then two patients received additional injections at the time
`points of months 4 and 6, respectively, and in the 0.5-mg/BRVO
`group, four patients, and then one, received additional injections at
`time points of months 4 and 6, respectively.
`
`Effect of ranibizumab on visual acuity
`In the CRVO group at the primary endpoint, the change from base-
`line (in the median number of letters read on an Early Treatment
`Diabetic Retinopathy Study chart) at 4 m was 17 and 14 in patients
`treated with 0.3 or 0.5 mg of ranibizumab, respectively (Figure 5a
`and b). All the patients in the 0.3-mg group and eight patients in
`the 0.5-mg group showed improvement in vision at the primary
`endpoint compared to baseline. One of the patients showed a
`reduction of 16 letters at the endpoint of 3 months, that was back
`to baseline at 4 months. The other patient showed a reduction of
`11 letters at the endpoint of 3 months that was judged to be due
`to progression of edema despite administration of ranibizumab;
`there was no evidence of ischemia or any other problem that
`could be attributed to the drug. The percentage of patients with
`
` clinically significant visual improvement defined as an improve-
`ment of ≥15 letters was 70% in the 0.3-mg group and 40% in the
`0.5-mg group.
`At the primary endpoint in patients with macular edema due
`to BRVO, the median change in visual acuity (VA) from baseline
`was 10 letters in the 0.3-mg group, and 18 in the 0.5-mg dose
`group (Figure 5c and d). One patient in the 0.5-mg dose group
`showed a reduction in VA of three letters at the primary endpoint,
`but all other patients showed improved vision. The percentage of
`patients with clinically significant visual improvement defined as
`an improvement of ≥15 letters was 40% in the 0.3-mg group and
`70% in the 0.5-mg group.
`
`Effect of duration of disease and patient’s age
`on visual outcome
`Figure 6 shows scatter plots of change from baseline in VA versus
`the patient’s age or duration of disease for patients with CRVO
`or BRVO treated with ranibizumab. There was no correlation
`between the amount of improvement in VA and duration of
`edema. There were four patients who had edema from CRVO for
`>2 years prior to starting injections of ranibizumab and three of
`them improved in VA by >15 letters. Three patients had edema
`from BRVO for >2 years at baseline and they showed improve-
`ments of 41, 21, and 14 letters in 3 months. Therefore, chronic
`edema from a vein occlusion does not preclude visual improve-
`ment as a result of intraocular injections of ranibizumab. There
`was no correlation between the patient’s age and visual improve-
`ment; improvement in VA of >15 letters occurred in two out of
`
`Molecular Therapy vol. 16 no. 4 april 2008
`
`793
`
`Mylan Exhibit 1036
`Mylan v. Regeneron, IPR2021-00881
`Page 3
`
`Joining Petitioner: Apotex
`
`

`

`Ranibizumab for Macular Edema in Vein Occlusions
`
`© The American Society of Gene Therapy
`
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`Figure 2 Excess foveal thickness at each visit from baseline (BL) to month 6 (M6) in patients with central retinal vein occlusion. The bars
`represent excess foveal thickness (central 1 mm retinal thickness—212 μm), which is a measure of the amount of macular edema, at BL just prior to
`injection of ranibizumab, and at day 7 (D7) and months 1 (M1), 2 (M2), 3 (M3), 4 (M4), and 6 (M6) for patients that received three injections of
`(a) 0.3 mg or (b) 0.5 mg of ranibizumab. The arrows show when injections were done (R, ranibizumab; B, bevacizumab; T, triamcinolone). Fairly rapid
`improvement in excess foveal thickness occurred in essentially all patients in the 0.5-mg dose group and most patients in the 0.3-mg group, but a few
`patients in the latter group showed small and/or delayed responses. Most patients showed recurrent edema 1–3 months after the last injection.
`showed elevation of blood pressure, thromboembolic events, or
`three patients with CRVO and three out of four patients with
`any other systemic problems. One patient, an 83-year-old male
`BRVO who were >80 years at baseline.
`with a history of pre-existent heart disease, died from a myocar-
`dial infarction 6 months after the last injection of ranibizumab;
`this was judged to be unrelated to the ranibizumab. As noted
`above, 38 out of 40 subjects showed improvement in VA at the
`primary endpoint compared to baseline and in the other two
`patients the reduction in vision was not felt to be attributable to
`ranibizumab.
`
`VEGF levels in aqueous
`The mean aqueous VEGF level at baseline was 39 pg/ml in 17
`patients with BRVO and 380 pg/ml in the 18 patients with CRVO
`for which measurements could be performed, a difference that
`was statistically significant (P = 0.001). There was an inverse cor-
`relation between baseline aqueous VEGF level and visual outcome
`in CRVO patients considered alone (P = 0.038) and for CRVO and
`BRVO patients considered together (P = 0.038); although BRVO
`patients considered alone did not reach statistical significance
`(Figure 7). There was no correlation of VEGF levels with excess
`foveal thickness at baseline or change in excess foveal thickness
`after treatment with ranibizumab.
`
`DISCUSSION
`Although this is an uncontrolled, open-label trial involving a rela-
`tively small number of patients, the results were very consistent
`among patients and suggest that intraocular injections of ranibi-
`zumab have a substantial effect on macular edema due to CRVO
`or BRVO. In both patient populations, the results were good with
`either 0.3 or 0.5 mg of ranibizumab and no clear differences could
`be discerned between the doses except that more patients seemed
`to have rapid improvements in center subfield thickness and more
`
`Safety
`Intraocular injections of ranibizumab were tolerated well
`with no inflammation or other problems. None of the patients
`
`794
`
`www.moleculartherapy.org vol. 16 no. 4 april 2008
`
`Mylan Exhibit 1036
`Mylan v. Regeneron, IPR2021-00881
`Page 4
`
`Joining Petitioner: Apotex
`
`

`

`© The American Society of Gene Therapy
`
`Ranibizumab for Macular Edema in Vein Occlusions
`
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`Figure 3 Cross sections through the fovea obtained by optical coherence tomography in patients with branch retinal vein occlusion. The hori-
`zontal cross sections at baseline (BL), day 7 (D7), month 1 (M1), month 2 (M2), and month 3 (M3, primary endpoint) are shown for five randomly
`selected patients of the ten patients treated with (a) 0.3 mg or (b) 0.5 mg of ranibizumab.
` ranibizumab, because the patient’s age being advanced at baseline
`had improvements that lasted for a month after the initial injection
`did not negatively impact the outcome.
`in the 0.5-mg groups compared to the 0.3-mg groups. However, at
`No drug-related adverse effects such as elevation of blood
`the 3-month primary endpoint, 1 month after the third injection,
`pressure, thromboembolic events, or any other systemic problems
`~90% of excess foveal thickness was eliminated in both patient
`were observed. This provides some preliminary data, suggesting
`populations with either dose. In addition, improvements from
`that intraocular injections of ranibizumab are well-tolerated in
`baseline in VA were large in both dose groups for patients with
`patients with retinal vein occlusions just as they are in patients
`macular edema due to CRVO or BRVO. These results are very
`with neovascular age-related macular degeneration.11,12
`encouraging and strongly support the performance of larger, more
`definitive studies.
`Measurement of VEGF levels in aqueous demonstrated a
`The Central Retinal Vein Occlusion Study was a large multi-
`higher mean level in patients with CRVO compared to patients
`center trial that investigated the effect of grid laser photocoagula-
`with BRVO. In patients with CRVO there was an inverse correla-
`tion in patients with macular edema due to CRVO.10 Although 69%
`tion with VEGF level at baseline and the visual outcome. Further
`work is needed to determine the predictive value of baseline aque-
`of patients in the treated group compared to 0% in the untreated
`ous levels of VEGF and to determine the range of VEGF levels that
`group showed reduction of fluorescein leakage in the macula at
`occur in other disease processes such as neovascular age-related
`the end of 1 year, there was no difference in the final VA (20/200
`in treated patients versus 20/160 in untreated patients). It has been
`macular degeneration and diabetic macular edema.
`felt that a possible explanation is that chronic edema due to CRVO
`In addition to grid laser therapy, several treatments have been
`leads to permanent visual loss. Our data demonstrate that visual
`tried in patients with macular edema due to CRVO including
`improvement is possible in some patients who have had edema
`the use of anticoagulants, fibrinolytics, steroids, acetozolamide,
`for >1 year and in some cases several years. In fact, there was no
`isovolemic hemodilution, surgically induced retinochoroidal
`inverse correlation between duration of edema at baseline and
`anastamoses or laser-induced retinochoroidal anastamoses, and
`improvement in VA after three injections of ranibizumab, sug-
`radial optic neurotomy. A recent meta-analysis of all published
`gesting that patients with chronic edema should not be excluded
`randomized clinical trials concluded that there is no convincing
`from treatment based solely upon the duration of edema. Our
` evidence that any of these treatments provide benefit.13 In con-
`data suggest that chronicity of edema and the relatively slow reso-
`trast, grid laser therapy provides modest benefit in patients with
`lution of edema after grid laser therapy are not likely to explain
`macular edema due to BRVO;6 after 3 years, patients treated with
`the poor visual results after grid laser therapy. Our data also sug-
`grid laser photocoagulation improved by 1.33 lines from base-
`gest that elderly patients with macular edema due to CRVO or
`line compared to an improvement of 0.23 lines in the control
`BRVO should not be excluded from treatment with intraocular
`group. There are several case series suggesting possible benefit
`
`Molecular Therapy vol. 16 no. 4 april 2008
`
`795
`
`Mylan Exhibit 1036
`Mylan v. Regeneron, IPR2021-00881
`Page 5
`
`Joining Petitioner: Apotex
`
`

`

`Ranibizumab for Macular Edema in Vein Occlusions
`
`© The American Society of Gene Therapy
`
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`(cid:18)(cid:36)(cid:24)(cid:25)(cid:34)(cid:34)(cid:1)(cid:33)(cid:25)(cid:35)(cid:28)(cid:32)(cid:23)(cid:30)(cid:1)(cid:35)(cid:27)(cid:28)(cid:24)(cid:29)(cid:32)(cid:25)(cid:34)(cid:34)(cid:1)(cid:2)(cid:28)(cid:32)(cid:1)(cid:1)(cid:31)(cid:3)
`
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