|Molecular Vision 2005;
Received 18 November 2004 | Accepted 23 May 2005 | Published 27 May 2005
Inhibition of retinal and choroidal neovascularization by a novel KDR kinase inhibitor
Fumi Kinose,1 Giuseppe Roscilli,2 Stefania
Lamartina,2 Kenneth D. Anderson,3 Fabio Bonelli,4
Stanley G. Spence,5 Gennaro Ciliberto,6 Thomas F.
Vogt,7 Daniel J. Holder,8
Catherine J. Thut1
(The first two authors contributed equally to this publication)
Departments of 1Ophthalmics Research, 3Medicinal Chemistry, 5Safety Assessment, 8Biometrics Research, Merck & Co., West Point, PA; Departments of 2Biochemistry, 4Pharmacology, and 6Molecular and Cellular Biology, IRBM, Merck & Co., Rome, Italy; 7Department of Molecular Profiling, Merck & Co., Rahway, NJ
Correspondence to: Catherine J. Thut, Department of Ophthalmics Research, Merck & Co., 770 Sumneytown Pike, WP26A-2000, West Point, PA, 19486; Phone: (215) 652-5728; FAX: (215) 993-5098; email: firstname.lastname@example.org
Purpose: Inhibition of vascular endothelial growth factor (VEGF) signaling has shown great promise for the treatment of ocular neovascular disease. Current anti-VEGF therapies in late-stage development, while efficacious, require dosing by frequent intravitreal injections that are inconvenient to patients. VEGF signaling inhibitors that demonstrate more convenient dosing regimens could lead to the improved treatment of neovascular diseases such as wet age related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). Here we describe the assessment of a KDR (VEGFR2) kinase inhibitor in two well-established models of ocular neovascularization following oral administration.
Methods: A novel KDR kinase inhibitor was dosed by oral gavage for 12 days at 0, 10, 30, or 100 mg/kg in an adult male Brown Norway rat laser induced choroidal neovascularization (CNV) model. The areas of CNV lesions were quantitated by fluorescence image analysis of FITC-dextran perfused animals. The kinase inhibitor was also assessed in a rat oxygen induced retinopathy (OIR) model in which neonatal rats were placed in an oxygen chamber that delivered alternating 24 h cycles of 50% and 10% oxygen for 14 days. After 14 days of oxygen treatment, the animals were returned to room air and dosed orally for 7 days with 0, 10, or 30 mg/kg kinase inhibitor. The extent of retinal neovascularization was assessed by counting pre-retinal neovascular nuclei on histological sections.
Results: At doses of 100 mg/kg, the KDR kinase inhibitor resulted in a 98% reduction in lesion size in the rat CNV model. 30 mg/kg doses of the inhibitor showed a 70% and 80% reduction in lesion size in the laser CNV and OIR models, respectively.
Conclusions: Oral dosing of the described KDR kinase inhibitor effectively inhibits neovascularization in two well-established animal models of ocular neovascularization. These data suggest that compounds of this class may prove to be useful for the treatment of a variety of ocular neovascular diseases using a convenient oral dosing regimen.
Despite current therapies, disorders of the ocular vasculature remain the leading causes of newly acquired blindness in the developed world. The two major diseases that affect the ocular vasculature are choroidal neovascularization (CNV), also called wet or exudative age related macular degeneration (AMD), and diabetic retinopathy. CNV accounts for the majority of new cases of blindness in persons over 50 years of age; 500,000 new cases are diagnosed each year. Photodynamic therapy is the primary treatment for wet AMD but is only modestly effective and indicated in the subset of patients with small, more superficially located neovascular lesions [1,2]. Diabetic retinopathy, on the other hand, may strike earlier in life and is the major cause of new blindness in working age adults. Panretinal laser photocoagulation (PRP) is a relatively effective treatment for the angiogenic form of the disease, termed proliferative diabetic retinopathy (PDR) [3,4]. PRP treatment serves to decrease retinal hypoxia (an angiogenic trigger) by destroying a large portion of the peripheral retinal neurons to prevent further angiogenesis and central vision loss. However, moderate visual disturbances such as loss of peripheral vision and changes in color perception are reported in about 15% of patients treated with PRP . Additional treatments that are more efficacious than photodynamic therapy and have fewer visual side effects than PRP are desirable and would lead to improved treatment of ocular neovascular diseases.
Recent efforts to develop improved treatments for ocular neovascular diseases have focused on pharmacologic therapies for directly inhibiting the growth and development of neovascular vessels [1,4]. While a large number of growth factor pathways have been implicated in angiogenesis, a variety of preclinical and clinical studies suggest that vascular endothelial growth factor (VEGF) is a central player in pathologic neovascularization both in the eye and elsewhere in the body . Elevated levels of VEGF mRNA and protein were demonstrated in ocular tissues and fluids from patients with both CNV [6-8] and PDR [7,9,10]. Furthermore, animal models that mimic aspects of these diseases, such as the rodent and primate laser CNV models [11,12] and the rodent oxygen induced retinopathy models [13,14], display increased VEGF levels as well. The central role played by VEGF in both neoplastic and ocular diseases has led to the development of a large number of VEGF pathway inhibitors including KDR antagonists, anti-VEGF antibodies and aptamers, and siRNAs to inhibit the expression of both Flt-1 (VEGFR1) and VEGF . To date, clinical trials to test the utility of anti-VEGF therapy in the eye have utilized localized delivery of anti-VEGF agents via frequent intravitreal injections (once every 4-6 weeks). A variety of other VEGF pathway inhibitors have not yet reached clinical trials and thus their route and frequency of dosing is not yet known.
VEGF signals through two tyrosine kinase receptors, Flt-1 (VEGFR1) and KDR (VEGFR2, Flk-1). VEGF binding to Flt-1 induces only weak phosphorylation of Flt-1 , and the signal transduction pathways involved are not yet well understood. Some evidence suggests that Flt-1 serves as a "decoy" receptor whose main function is to regulate the amount of free VEGF in the extracellular space [17,18]. VEGF binding to KDR, on the other hand, causes receptor dimerization and autophosphorylation on several tyrosine residues in the KDR cytoplasmic domain . Activation of KDR in this manner leads to signaling through a variety of pathways including Raf-Mek-Erk and PI-3 kinase-Akt [19-22]. The former is involved in endothelial cell proliferation, while the latter induces the expression of anti-apoptotic genes thought to protect endothelial cells in nascent vessels formed during embryonic development and in pathologic conditions .
While VEGF clearly plays a central role in the development of neovascular diseases, other growth factor pathways, including those that signal through additional receptor tyrosine kinases such as platelet derived growth factor receptor (PDGFR), fibroblast growth factor receptors (FGFRs), Flt-1, and Flt-4, have been implicated in neovascularization and ocular disease [24-26]. Some studies have suggested that inhibition of VEGF signaling alone is sufficient to cause a decrease in angiogenesis, though others have demonstrated more potent decreases in angiogenesis with the use of anti-angiogenic agents that inhibit multiple tyrosine kinase receptors [27,28]. Thus, pharmacologic agents that inhibit multiple angiogenic tyrosine kinases may be a more desirable approach to the treatment of ocular neovascular disease.
A series of novel indolyl quinolinone compounds with activity against KDR and several closely related tyrosine kinases and with good oral bioavailability have recently been reported . One of these compounds, 3-(5-[2-[(2-methoxyethyl)(methyl)amino]ethoxy]-1H-indol-2-yl)-2(1H)-quinolinone (Compound A; Figure 1), has been shown to effectively inhibit tumor angiogenesis and growth in a murine tumor model . In this study, we show that this same compound is effective in suppressing angiogenesis in two animal models of ocular neovascular disease, rat laser CNV and rat OIR (oxygen-induced retinopathy).
KDR kinase inhibitor
The synthesis of Compound A is described in detail elsewhere [29,30]. Dosing solutions were made by dissolving the appropriate amount of Compound A (free base weight) in 0.5% carboxymethylcellulose (Sigma, St. Louis, MO).
Experiments were conducted in accordance with the standards established by the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals) and the Merck & Co. Institutional Animal Care and Use Committee.
Rat laser CNV model
The rat laser CNV model was performed essentially as described by Edelman and Castro . Male Brown Norway rats (10-12 weeks in age, Charles River Labs, Raleigh, NC) were anesthetized with 70 mg/kg ketamine and 7 mg/kg xylazine (Fort Dodge Animal Health, Fort Dodge, IA) and their pupils dilated with 1 drop of 1% tropicamide (Alcon Labs, Ft. Worth, TX). Using a drop of an ocular lubricating solution (Celluvisc, Allergan Inc., Irvine, CA) and a coverslip as a contact lens, the fundus was viewed using a Nidek SL-1600 slit lamp (Nidek, Gamagori, Japan), and three argon laser lesions were produced with a Nidek ADC-8000 laser in a circle around the optic disc (488-514 nm, 80 mW, 100 ms, 0.1 mm spot size). All laser lesions resulted in a "vapor bubble" indicating rupture of Bruch's membrane. Animals that developed large hemorrhages in response to laser treatment were excluded from the study and replaced. Animals were divided into 4 groups of 6 and dosed once daily by oral gavage starting on the day of laser (day 0) with Compound A at 0, 10, 30, or 100 mg/kg using 0.5% methylcellulose as a vehicle. The treatment group size was chosen based on statistical analysis of the lesion size variances we typically observe in the rat laser CNV model. Based on these calculations, we had >90% confidence that a sample size of 6 animals would allow us to distinguish a compound that results in about 40% reduction in lesion size compared to vehicle control. On day 11, a blood sample was drawn from the tail vein at 1 h and 4 h after dosing (for some of the animals it was difficult to draw blood from the tail vein and for these animals 1 and/or 4 h timepoints were not collected). On day 12, the animals were deeply anesthetized with Nembutal (Abbott Laboratories, North Chicago, IL), an intracardiac blood sample was drawn, and the animals were perfused with 50 ml lactated Ringer's solution (Abbott Laboratories, North Chicago, IL) followed by 20 ml of 5 mg/ml FITC-dextran (MW 2x106; Sigma, St. Louis, MO) +10% gelatin (75 bloom; Sigma, St. Louis, MO) dissolved in lactated Ringer's solution. The eyes were enucleated and fixed overnight in 10% phosphate buffered formalin (Fisher Scientific, Fair Lawn, NJ) at 4 °C.
Adult rat PK studies
Protein was precipitated from the plasma samples using acetonitrile, and compound levels were measured in the liquid phase using liquid chromatography (10% acetonitrile/0.1% formic acid to 90% acetonitrile/0.1% formic acid gradient) followed by mass spectrometry. Instrumentation consisted of a LEAP Technologies autosampler and an Agilent Technologies HP 1100 HPLC with a Phenomenex Prodigy ODS3 100A 50x2 mm column interfaced via a Sciex Turbo Ion Spray Source to a Sciex API 2000 Mass Spectrometer. Plasma compound concentrations were calculated from a standard curve.
Assessment of choroidal neovascularization by fluorescence microscopy
The RPE-choroid-sclera from fixed laser CNV eyes was mounted on a microscope slide by cutting around the equator of the eye, gently removing the anterior half of the globe, the vitreous and the neural retina, then making four radial cuts in the posterior half to allow the tissue to be mounted flat and covered with Fluoromount G (Electron Microscopy Sciences, Hatfield, PA) and a coverslip. Laser lesions were visualized by fluorescence microscopy using a GPF filter (Nikon Eclipse TE300, Nikon, Inc., Melville, NY). Digital images of the laser lesions were captured and areas of neovasculature were measured using Image-Pro Plus software (Media Cybernetics, Silver Springs, MD). All 6 lesions were imaged and quantitated for all animals.
Neonatal rat tolerability and PK studies
Forty-eight postnatal day 14 Sprague Dawley rats were divided into 4 groups of 12 and administered 0, 10, 30, or 100 mg/kg Compound A by oral gavage for 7 days. Body weight measurements were made daily. The 100 mg/kg dose group was euthanized on days 17-19 due to mortality and weight loss. The remainder of the animals were euthanized on day 21 with isoflurane (Vedco Inc., St. Joseph, MO) and plasma samples collected for drug level analysis. Protein was precipitated from the plasma samples using acetonitrile, and compound levels were measured in the remaining liquid phase using liquid chromatography (5% acetonitrile/10% methanol/0.1% formic acid to 95% acetonitrile/0.5% methanol/0.1% formic acid gradient) followed by mass spectrometry. Instrumentation consisted of an HTS PAL CTC autosampler, an Agilent Technologies HP 1100 HPLC and an Xterra C18 4.6x50 mm column interfaced via a Sciex Turbo Spray Source to a Sciex API 3000 Mass Spectrometer. Plasma compound concentrations were calculated from a standard curve.
Rat oxygen-induced retinopathy model
Within 2 h of birth, 53 newborn Sprague-Dawley rats and their nursing dams were placed in a controlled atmosphere chamber and exposed to 24 h alternating cycles of hyperoxia (50% O2) and hypoxia (10% O2) as described by Penn et al. . At postnatal day 14, the rats were returned to room atmosphere for 7 days to allow the development of retinal neovascularization. At the time the rats were transferred to room air, each litter was divided into 3 dosing groups: 0 mg/kg (n=21), 10 mg/kg (n=13), and 30 mg/kg (n=19) Compound A. The neonatal rats were dosed once daily by oral gavage for 7 days.
Visualization of retinal neovascularization by fluorescence angiography
Five animals from each dosing group were anesthetized with 50 mg/kg ketamine and 6 mg/kg xylazine (Fort Dodge Animal Health, Fort Dodge, IA) and then perfused via the left ventricle with 1 ml of 50 mg/ml FITC-dextran (MW 2x106; Sigma) dissolved in phosphate buffered saline. The animals were then sacrificed, their eyes enucleated and retinas flat mounted as described . The retinal vasculature was visualized by fluorescence microscopy.
Quantitative assessment of retinal neovascularization by analysis of serial retinal sections
Quantitative analysis of retinal neovascularization was performed by counting the number of endothelial cell nuclei extending beyond the inner limiting membrane and protruding into the vitreous in histological sections of eyes . Briefly, the rats were euthanized by CO2 inhalation, and one eye from each rat was enucleated, fixed in 4% paraformaldehyde, alcohol dehydrated and embedded in paraffin. Sections were stained with hematoxylin and eosin. An average of 360 readable 6 μm sections were cut beginning as soon as the ciliary plexus was detectable, continuing once it disappeared and ending when it reappeared on the far side of the eye. The number of nuclei crossing the inner limiting membrane was counted on every other section (about 180 sections per eye) by two independent and masked observers. 16, 8, and 14 eyes were analyzed for the 0, 10, and 30 mg/kg groups, respectively. For each animal, the average number of preretinal nuclei per section was calculated. The treatment group size was chosen based on statistical analysis of the variances in preretinal nuclei counts we typically observe in the rat oxygen-induced retinopathy model. Based on these calculations, we had 90% confidence that a sample size of 12 animals would allow us to distinguish a compound that results in about 80% reduction in lesion size compared to vehicle control.
For the rat laser CNV model, a one way analysis of variance (ANOVA) model was used to assess the effects of drug treatment. Differences between each dose group and the vehicle group were assessed using a Student's t-test and the error estimate from the ANOVA model. Since variability tended to increase with lesion size, the log transformation was used to homogenize the variance. Individual lesion areas were log transformed and averaged for each rat prior to fitting the ANOVA model. Because the experimental design was completely balanced, this method yields p values and confidence intervals that are identical to those produced by a repeated measured analysis. For the rat OIR model, the effects of drug treatment were assessed using Student's t-test and the pooled error estimate from a one way ANOVA. A square root transformation of the average number of nuclei/section/rat was used to homogenize the variance prior to fitting the ANOVA model.
Oral dosing of Compound A robustly decreases neovascularization in a rat experimental CNV model
An established rat model  was used to assess the ability of Compound A to inhibit choroidal neovascularization. Groups of 6 experimental animals were administered 0, 10, 30, or 100 mg/kg Compound A once daily by oral gavage for 12 days. Lesion size (in μm2) was assessed on day 12. As shown in Figure 2A, robust choroidal neovascular lesions formed in lasered, vehicle treated rats. Rats treated with 10 mg/kg Compound A also displayed large, well formed lesions (Figure 2B). Lesions in rats treated with either 30 or 100 mg/kg Compound A, however, appeared in aggregate to have many fewer vessels (Figure 2C,D). Quantitation of the lesions by computer aided image analysis confirmed these qualitative assessments and showed a statistically significant 98% reduction in lesion size in the 100 mg/kg treatment group and a 70% reduction in the 30 mg/kg group as compared to vehicle controls (Figure 2E). Although we observed a 20% reduction in lesion size in the 10 mg/kg group compared to vehicle, this difference did not achieve statistical significance (Figure 2E). Body weight measurements over the 12 days of dosing did not reveal statistically significant weight changes as compared to vehicle treated controls at any of the doses tested (data not shown).
Plasma samples collected at 1, 4, and 24 h after compound dosing show dose proportional plasma levels of Compound A. At the 24 h timepoint, the 100 mg/kg dosed animals showed 722-314 nM of Compound A, a plasma level predicted to be in excess of its IC90 for KDR (about 190 nM, unpublished data) while the 30 mg/kg dose showed a near IC50 plasma level for KDR (about 20 nM ) at 24 h (Table 1). Plasma levels of Compound A were below the limit of quantitation (<13 nM) at 24 h in the 10 mg/kg treated group.
Oral dosing of Compound A decreases retinal neovascularization in a rat oxygen-induced retinopathy model
Hypoxia induces the expression of VEGF and plays a key role in the development and progression of PDR. While there are currently no well established animal models of true proliferative diabetic retinopathy, oxygen induced rodent models of retinopathy are often used to mimic the hypoxic trigger of PDR. Because neonatal animals are more sensitive to a variety of drugs than adults, a pilot study was performed to determine the maximum tolerated dose in neonatal rats dosed with Compound A once daily from postnatal day 14-21. After 1 week of oral dosing at 10 or 30 mg/kg Compound A, the neonatal rats appeared generally healthy and displayed either a 0% or a 15% decrease in body weight, respectively (data not shown). Unlike adult rats, 100 mg/kg was poorly tolerated by neonatal rats as evidenced by excessive weight loss (data not shown). Blood samples were collected on postnatal day 21 at 1 h and 24 h after the last dose for plasma drug level analysis (Table 2) and showed the levels were dose proportional at 1 h. At 24 h, the plasma levels were below the limit of quantitation (<2 nM).
Based on the results of the tolerability study, 10 and 30 mg/kg doses of the kinase inhibitor were chosen for testing in the rat OIR model. Three groups of neonatal rats previously exposed to a controlled oxygen environment were dosed daily with 0, 10, or 30 mg/kg Compound A for 7 days. Fluorescence microscopy of retinas from fluorescein-dextran perfused animals revealed that treatment with 30 mg/kg (Figure 3B) but not 10 mg/kg (not shown) Compound A caused a reduction in the area of retinal vascularization as compared to vehicle treated control (Figure 3A). Oxygen induced retinal neovascularization in the rat OIR model is also characterized by the development of preretinal vessels (Figure 3C,D). Quantitative measurements of preretinal neovascularization between the treatment groups were made by counting the number of nuclei lying above the inner limiting membrane in standard histological sections. These measurements revealed a statistically significant, 80% reduction in preretinal neovascular nuclei in the 30 mg/kg kinase inhibitor treatment group as compared to vehicle treatment (Figure 3E). In agreement with the fluorescence angiography data, there was not a statistically significant difference in neovascular nuclei between the 10 mg/kg dose group and vehicle groups. The general health and development of the animals were monitored by daily body weight measurements and gave similar results to those obtained in the preliminary tolerability studies (data not shown).
Our studies demonstrate that oral dosing of Compound A potently inhibits neovascularization in animal models of retinal and choroidal neovascularization. The rat laser CNV and OIR models mimic important aspects of wet AMD and PDR, respectively, suggesting that this kinase inhibitor may be useful in the treatment of these prevalent and debilitating ocular diseases. In the laser CNV model, doses of 100 mg/kg showed nearly complete inhibition of choroidal neovascular growth. Dose limiting toxicities observed in neonatal but not adult rats precluded testing the kinase inhibitor at 100 mg/kg in the OIR model. The 30 mg/kg dose decreased lesion size by 70% in the CNV model and reduced the number of neovascular nuclei in the OIR model by nearly 80%. The lack of maximal inhibition at the lower doses suggests that it is necessary to maintain plasma levels greater than the IC50 for KDR kinase inhibition for a significant portion of each day. Future studies may include the assessment of Compound A's effect on neovascularization in animals of different ages and strains to address potential differences in response.
While Compound A was originally developed as a KDR kinase inhibitor, it also inhibits several other receptor tyrosine kinases including PDGFR, c-kit, Flt-4 (VEGFR3) and c-fms with IC50's for these kinases in the range of 0.8-2.0 nM . Significant inhibition of these receptors is expected at both the 30 and 100 mg/kg doses. At the 100 mg/kg dose, Flt-1, Src and FGFR1 and 2 signaling would also be affected. PDGF and Flt-4 have both been implicated in ocular neovascularization [35,36]; Flt-4 is expressed in endothelial cells [36,37] and PDGFR on pericyte cells [38,39] that both contribute to capillary structure. C-kit and c-fms are not expressed in growing blood vessels but instead are expressed in circulating endothelial cell precursors and macrophages, respectively. There is mounting evidence to suggest that these circulating cell types are intimately involved in the regulation of vessel development as the sources of either secreted angiogenic cytokines or nascent endothelial cells that can be recruited to growing blood vessels [40,41]. The ability of Compound A to inhibit PDGFR is of particular interest because several recent studies have shown that simultaneous inhibition of both KDR and PDGFR may not only halt neovascularization but may also cause limited regression of newly formed vessels that have not yet developed stable endothelial cell:pericyte interactions [27,28]. For these reasons, the ability of the described kinase inhibitor to antagonize VEGF and other angiogenic signals suggests that it may more potently reduce the neovascularization associated with wet AMD and PDR than VEGF specific therapies.
Delivery of small molecules and biologics to the eye continues to be a challenge for the development of safe, efficacious treatments for retinal diseases. The accessibility and relative isolation of the eye has encouraged the use of intravitreal injections for compound delivery. While this approach has allowed the successful delivery of anti-angiogenic agents to the eye, frequent intravitreal injections are unpleasant for patients and carry with them the risk of intraocular infection. Therefore, more convenient routes of delivery are desirable. The oral bioavailability of this class of kinase inhibitors could allow a more convenient dosing regimen in man. However, concerns have been raised that the long term use of a systemic anti-angiogenic agent may carry cardiovascular risks for the elderly and diabetic patients in whom the ability to form new vessels may be desirable after vascular events such as heart attack and stroke. There is also preclinical evidence for a role for VEGF signaling in neurogenesis and neuroprotection . Long term safety studies with compounds of this class will be required to determine whether these compounds are suitable for systemic administration in AMD and diabetic patients. In addition, alternative means of infrequent, localized ocular delivery that would avoid significant systemic exposure to the compound are being actively pursued. Assuming that a safe and efficient dosing regimen and delivery system can be developed, kinase inhibitors such as Compound A show great promise for use as therapeutics in the treatment of ocular neovascular disorders.
We thank Ernesta Dammassa, Jill Wiler Maxwell and Robert Breese for their precious technical assistance, and Konstantin Petrukhin, George Hartman, and Charles Tressler for critical reading of the manuscript.
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