KEY FEATURES

Early events in the development of diabetic retinopathy include the loss of retinal pericytes, thickening of the capillary basement membrane, and increased leukocyte adhesion to retinal endothelial cells. Elevated permeability of retinal endothelial cells leading to macular edema is caused by increased expression of vascular endothelial growth factor (VEGF), which has been found in the vitreous fluid of patients with diabeticretinopathyevenattheearlystagesofthedisease.1–3 Proliferative diabetic retinopathy is associated with neovascularization, which is most likely the result of deregulated proliferation and migration of retinal endothelial cells induced by growth factors like VEGF, basic fibroblast growth factor, or other stimulators.2,4 At least some of these processes are directly or indirectly associated with the activation of serine/threonine-specific protein kinase C (PKC) and its isoenzymes.5–9 PKC is therefore considered a valuable target molecule for therapeutic intervention (Figure 39.1).5–7,10–12

INTRODUCTION TO PROTEIN KINASE C

PROTEIN KINASE C FAMILY

Protein kinases transfer phosphate groups to either serine/threonine residues (e.g., protein kinaseA, PKC, mitogen-activated protein kinases) or to tyrosine residues (e.g., growth factor receptor kinases) of the target protein, leading to its activation or in some cases to its inactivation. The PKC family comprises several different members (PKCα, βI and II, γ, δ, ε, η, θ, ζ, ι/λ) which differ in their structure, cofactor requirement, and substrate specificity7,11 (Table 39.1 and Figure 39.2). These kinases, of which some are tissue-specifically expressed, are usually located in the cytoplasm. Activation of PKC isoenzymes (α, βI and II, δ, and ε) is usually accompanied by translocalization of the kinase activity from the cytosol to the plasma membrane within minutes in microvascular (retinal) and macrovascular endothelial cells after stimulation with high glucose, VEGF, or phorbol-12-myristate-13-acetate (PMA).13,14 Whether this short-term effect becomes permanent after long-term treatment of the cells with PMA or VEGF remains to be shown. Each PKC consists of a single polypeptide chain derived from a single gene, except for PKCβ I and II, which are alternative splice variants. PKC members which function as intracellular signal transduction systems for several cytokines and hormones, can be divided into three subgroups (Table 39.1 and Figure 39.2):

1. Activation of conventional or classical PKCs (cPKC: α, βI and II, γ) depends on Ca2+ ions and adenosine triphosphate (ATP). They can be activated by diacylglycerol or phorbol esters like PMA.

2.  Novel PKCs (nPKC: δ, ε, θ), which can also be activated by diacylglycerol or PMA, are independent of Ca2+ ions.

3.  PKCζ and PKCι/λ belong to the group of atypical PKCs (aPKCs) which are insensitive to diacylglycerol or PMA but can be activated by phosphatidylserine.

PKCη (PKD1) and PKCµ (PKD3) form a fourth subgroup of PKC isoenzymes, also known as the protein kinase D family, and are not discussed in this chapter.

EFFECTS OF ACTIVATED PKC

It is well known that hyperglycemia leads to elevated levels of diacylglycerol, which is a potent activator of PKC.15–17 It also results in the deregulation of several cellular processes in which PKC isoenzymes are involved, including those which lead to inhibition of Na-K ATPase, increasing permeability, and stimulation of proliferation in different cell types.9,18,19 Expression of several genes (cytokines, growth factors, nitric oxide synthase, extracellular matrix proteins) regulated by PKC depends on stimulation of mitogen-activated protein kinase and is mainly mediated through the transcription factors NFκb and AP-1 in retinal and other cells. PKC stimulation by PMA in macrovascular endothelial cells leads directly to synthesis of VEGF mRNA.20 Whether VEGF expression can be directly induced by activated PKC in retinal endothelial cells remains to be demonstrated, but at least in retinal pericytes expression of VEGF can be activated by stimulation of PKC.9,21

In vitro studies using cell culture models indicate that activation of PKC by PMA or high glucose also increases the permeability of various cell types, including (retinal) endothelial cells.19 The pathway of paracellular signaling in both endothelial and epithelial cells is regulated at tight junctions which consist of transmembrane proteins like occludin and claudins as well as membrane-associated proteins like zonula occludens-1.22 The composition of complexes containing these proteins determines the rate of transition of molecules through tight junctions, and their reorganization, including delocalization of its components, most likely causes the alteration of endothelial permeability in diabetic retinopathy. There is some evidence that phosphorylation of the tight junction protein occludin by PKCβ and/or PKCδ within 15 minutes of VEGF165 treatment influences its reversible translocalization from the plasma membrane to the cytoplasm in retinal endothelial cells, although this seems to be only a temporary effect.23,24 Expression of endothelin, which controls retinal blood flow, is also induced by PKCβ in the retina of diabetic animals as well as in retinal pericytes after cultivation in medium containing high glucose.25,26

PHARMACOLOGY OF RUBOXISTAURIN

Inhibition of PKC, especially of the β-isoform, seems to be a promising approach to treat diabetes-associated microvascular complications. This chapter discusses primarily the characteristics of ruboxistaurin, which specifically inhibits PKCβ, since the nonspecific PKC-inhibitor PKC412 showed severe adverse effects in treated individuals and is no longer under investigation.27 Ruboxistaurin mesylate (compound identifier LY333531, Eli Lilly, Figure 39.3) is a macrocyclic bisindolylmaleimide compound that specifically inhibits the β-isoform of PKC.28,29 As a competitive inhibitor for ATP, LY333531 inhibited isolated enzymes PKCβI and βII with a half-maximal inhibitory constant of 4.5 nM and 5.9 nM, respectively, whereas inhibition of other PKC isoforms required 250 times higher concentrations.29 There is strong evidence that CYP3A4 is the primary cytochrome P450 enzyme responsible for the metabolism of ruboxistaurin to its main equipotent metabolite, N-desmethyl ruboxistaurin (LY333522; Figure 39.3).30 The half-life of ruboxistaurin which can be orally administered is approximately 9 hours and that of its metabolite 16 hours, therefore allowing once-daily dosing. Studies showed that the primary excretion route in humans for these substances was fecal with renal elimination playing a minor role.31

Ruboxistaurin Inhibitor: C Kinase• 39Proteinchapter

Figure 39.1  Proposed central role of protein kinase Cβ (PKCβ) in the pathogenesis of diabetic retinopathy. Inhibition of PKCβ can affect the progression of diabetic retinopathy at different stages. Synthesis of diacylglycerol (DAG) and advanced glycation endproducts (AGE) is stimulated by hyperglycemia, resulting in the activation of PKC(β). As a consequence, vascular endothelial growth factor (VEGF) expression is stimulated in some cells, which in turn also activates PKC, leading to vascular leakage and neovascularization seen in diabetic retinopathy. These processes may be influenced by PKCβ inhibition. + stimulation of the process by PKC (activation); – inhibition of the process by PKC inhibition.

MECHANISM OF ACTION OF

PKC INHIBITORS

Several studies using animal models have indeed shown that PKC inhibitors can counteract cellular processes activated by PKC: Intravitreal injection of VEGF at clinically observed concentrations rapidly activated PKC in the retina in an animal model and led to a more than threefold increase in retinal vasopermeability. Intravitreal or oral administration of a PKCβ inhibitor almost completely reverted this VEGF-induced permeability.32 In addition, LY333531 prevented diabetes-induced retinal vascular leakage and retinal neovascularization in a mouse model for diabetes type 2.33 Treatment of diabetic rats with ruboxistaurin resulted in an amelioration of the retinal blood flow in a dose-responsive manner in parallel with inhibition of the retinal PKC activity.28 Ruboxistaurin also reduced the VEGF-induced blood– retinal barrier breakdown and neovascularization in an animal model. Thereby, the inhibitor abolished both VEGF-induced PKC activation and endothelial cell proliferation, with VEGF’s mitogenic effect being inhibited by ruboxistaurin in a concentration-dependent manner.32 This inhibitor also effectively inhibited preretinal and optic nerve head neovascularization in a porcine model of branch retinal vein occlusion without any apparent systemic toxicity.34 In this case, the ameliorative effect seemed to be a result of the disruption of the intracellular signal cascade activated by VEGF and other angiogenic growth factors by targeting one of its key components.

EFFECT OF RUBOXISTAURIN IN HUMAN NONOCULAR DISEASES

Since hypergylcemia results in disordered skin microvascular blood flow – possibly through overactivation of PKCβ – the effect of ruboxistaurin on neurovascular function was tested in patients with diabetic peripheral neuropathy. In a double-masked, placebocontrolled study a small number of patients (20 per group) were treated with placebo or 32 mg/day ruboxistaurin over 6 months.35 A significant increase of the endothelium-dependent and C-fiber-mediated skin microvascular blood flow at the distal calf as well as reduced sensory

symptoms were observed in the ruboxstaurin-treated group. In another clinical trial, 250 patients with diabetic peripheral neuropathy were treated with placebo, 32 or 64 mg/day ruboxistaurin over 1 year. However, no significant improvement in abnormal measurable vibration detection threshold was seen in the two groups treated with ruboxistaurin compared to the placebo group.36 Inhibition of PKCβ by ruboxistaurin also improved kidney disease in animal models.28,33 Therefore, patients with diabetes type 2-associated nephropathy were treated with 32 mg/day ruboxistaurin in a placebo-controlled pilot study over 1 year. However, no statistically significant changes were observed between the ruboxistaurinand the placebo-treated group with regard to albuminuria and estimated glomerular filtration rate, which might be due to the small number of individuals.37

Use of PKC InhibITORS in the treaTMENT of diabETIC macuLAR edemA and diabETIC retiNOPATHY

Treatment of DME patients for 3 months with a multitargeting kinase inhibitor PKC412, which also acts as a nonspecific PKC inhibitor, led to a reduction of retinal thickening as evaluated by optical coherence tomography. However the systemic applicability of this nonselective compound was limited by substantial gastrointestinal side-effects and other dose-related signs of low tolerability, e.g., disturbed glycemic control and liver toxicity.27 On the other hand, treatment with ruboxistaurin mesylate (4 and 32 mg/day), which selectively inhibits the PKCβ isoform, can reduce the retinal vascular leakage in eyes that have DME and markedly elevated leakage, suggesting that this treatment may be most effective in patients with severe forms of macular edema.38 In patients receiving 16 mg ruboxistaurin twice daily, the diabetes-induced increase in retinal circulation time was improved. A linear correlation between the dose of ruboxistaurin and its effect on retinal circulation time was observed as well as with retinal blood flow.39

EFFICACY OF RUBOXISTAURIN IN THE TREATMENT OF DIABETIC RETINOPATHY

In a multicenter, double-masked, randomized, and placebo-controlled study (PKC-DRS study) the safety and efficacy of orally administered ruboxistaurin were evaluated in subjects with moderately severe to very severe nonproliferative diabetic retinopathy.40 A total of 252 subjects received placebo or ruboxistaurin (8, 16, or 32 mg/day) for 36–46 months. Patients had an ETDRS retinopathy severity level between 47B and 53E inclusive, an ETDRS visual acuity of 20/125 or better, and no history of scatter photocoagulation. Efficacy measurements included progression of diabetic retinopathy, moderate visual loss, and sustained moderate visual loss. Compared with placebo, 32 mg/day ruboxistaurin was weakly associated with a delayed occurrence of moderate visual loss (P = 0.038) and sustained moderate visual loss (P = 0.226). This was evident only in eyes with definite DME at baseline (P = 0.017). As a result of a multivariable Cox proportional hazard analysis, 32 mg/ day ruboxistaurin significantly reduced the risk of moderate visual loss compared with placebo (P = 0.012).40 The beneficial effect of ruboxistaurin on moderate visual loss might be due to improved retinal cell viability resulting from PKCβ inhibition, leading to a greater resistance of retinal vascular and neural cells to pathologic stress of hyperglycemia and changes in the hemodynamics of blood flow. Further multicenter trials (e.g., PKC-DMES study) investigated whether ruboxistaurin can reduce the progression of DME and diabetic retinopathy: In a multicenter, double-masked, randomized, placebo-controlled study 686 patients who had DME farther than 300 µm from the center of the macula at baseline were treated with either ruboxistaurin (4, 16, or 32 mg/day) or placebo for 30 months. The primary outcome was progression to sight-threatening DME or application of focal/grid photocoagulation for DME. Although the progression to this primary

Diseases Retinal in Mechanisms and Drugs • 4 section

Ruboxistaurin Inhibitor: C Kinase• 39Proteinchapter

nephropathy in type 2 diabetes. Diabetes Care 2005;28:2686–2690.

38.Strom C, Sander B, Klemp K, et al. Effect of Ruboxistaurin on blood–retinal barrier permeability in relation to severity of leakage in diabetic macular edema. Invest Ophthalmol Vis Sci 2005;46:3855–3858.

39.Aiello LP, Clermont A, Arora V, et al. Inhibition of PKCβ by oral administration of Ruboxistaurin is well tolerated and ameliorates diabetes-induced retinal hemodynamic abnormalities in patients. Invest Ophthalmol Vis Sci 2006;47:86–92.

40.The PKC-DRS Study Group. The effect of ruboxistaurin on visual loss in

patients with moderately severe to very severe nonproliferative diabetic retinopathy. Diabetes 2005;54:2188–2197.

41.The PKC-DMES Study Group. Effect of ruboxistaurin in patients with diabetic macular edema. Arch Ophthalmol 2007;125:318–324.

42.Yeo KP, Lowe SL, Lim MT, et al. Pharmacokinetics of ruboxistaurin are significantly altered by rifampicin-mediated CYP3A4 induction. Br J Clin Pharmacol 2005;61:200–210.

Diseases Retinal in Mechanisms and Drugs • 4 section