SECTION IV • MECHANISMS OF HYPERGLYCAEMIA INDUCED VASCULAR DYSFUNCTION 214 Fig. 24.1 Structure of ruboxistaurin, a macrocyclic bis-indolylmaleimide, which is an orally active, selective PKC-β inhibitor. N(CH 3 ) 2 H N O O NN O Table 24.1 Tabulated IC 50 values (i.e. concentrations in nM required to achieve 50% inhibition of enzyme activity) for ruboxistaurin and the non-specific PKC inhibitor, staurosporine, with respect to each PKC isoform and related intracellular kinases. Adapted from Science 1996; 472: 728–731. Tabulated IC 50 values for ruboxistaurin IC 50 (nM) Kinase ruboxistaurin Staurosporine PKC-α 360 45 PKC-β 1 4.7 23 PKC-β 2 5.9 19 PKC-γ 300 110 PKC-δ 250 28 PKC-ε 600 18 PKC-ζ >10 5 >1.5 × 10 3 PKC-η 52 5 Cyclic AMP kinase >10 5 100 Ca 2+ -calmodulin kinase 8 × 10 3 4 Casein kinase >10 5 1.4 × 10 4 Src tyrosine kinase >10 5 1 new vessel formation. Thus, blocking VPF-mediated retinal permeability is a prime target for therapeutic amelioration of diabetic maculopathy. Studies in rats have clearly shown that intravitreal administration of VPF increases vitreous fluorescein leakage, and that pretreatment of these animals for one week with ruboxistaurin 25 mg/kg/day via oral administrattion ame- liorated VPF and phorbol ester-induced vitreous fluorescein leakage (Fig. 24.2). Furthermore, whereas control rats showed a two-fold increase in vitre- ous fluorescein leakage after intravitreal VPF administration, rats pretreated with the PKC-β inhibitor showed no difference in basal vitreous fluorescein leakage but there was a 96% reduction in VPF-induced vitreous fluorescein leakage (Fig. 24.2). Increased retinal permeability is a hallmark of neovascularization within the diabetic eye, as well as being a sight-threatening pathological entity even in the absence of new vessel formation. These experimental data have shown that oral administration of ruboxistaurin is well tolerated and considerably CHAPTER 24 • EXPERIMENTAL PHARMACOLOGY USING ISOFORM-SELECTIVE PKC INHIBITORS 215 Fig. 24.2 Oral administration of the PKC-β inhibitor, ruboxistaurin, to normal rats prevents the increase in vitreous fluorescein leakage following intravitreal injection of VPF. Adapted from Diabetes 1997; 46: 1473–1480. Vitreous fluorescein leakage (arbitrary units) 20 10 0 0 0 2 0 0 25 2 25 VPF (ng/eye) PKC-β inhibitor (mg/kg rat/day) P=0.015 P=0.043 SECTION IV • MECHANISMS OF HYPERGLYCAEMIA INDUCED VASCULAR DYSFUNCTION 216 attenuates VPF-mediated retinal permeability. Furthermore, diabetes is char- acterized by an increase in retinal mean circulation time (MCT), and oral treatment with ruboxistaurin for two weeks in STZ-diabetic rats reduced reti- nal MCT, as measured by video fluorescein angiography (Fig. 24.3). This experimental data has now been confirmed in phase II clinical trials in which ruboxistaurin administration for one month produced significant improve- ments in retinal blood flow and MCT among 27 diabetic patients (chapter 25). Larger, multicentre clinical trials are in progress. Fig. 24.3 Effect of oral dosing with ruboxistaurin on renal and retinal vascular function in non-diabetic (●) and STZ-diabetic (●) rats. Untreated diabetic animals show increases in glomerular filtration rate (GFR), renal filtration fraction (GFR corrected for renal plasma flow, RPF), urinary albumin excretion rate (AER) and retinal mean circulation time (MCT). Oral treatment with ruboxistaurin 0.1–10 mg/kg/day ameliorated these renal and retinal haemodynamic abnormalities. Science 1996; 272: 728–731. 0 0.1 1.0 10 0 0.1 1.0 10 0 Dose of ruboxistaurin (mg/kg) Dose of ruboxistaurin (mg/kg) 1000 0.1 1.0 10 GFR (ml/min) Filtration fraction (GFR/RPF) Urinary AER (mg/day) MCT (s) 0 20 2 1.5 0.5 0 1 15 10 5 0 2 4 + + + + + + + + + * * * § § § § § 6 (a) (b) (c) (d) 0.5 0.4 0.3 0.2 0.1 0 Further experimental studies have shown that diabetes-induced reduc- tions in Na +/ K + -ATPase and Ca 2± -ATPase in the retina are mediated, in part, via PKC-β activation. Oral administration of ruboxistaurin normalizes Na + /K + -ATPase activity in retinal microvessels (Fig. 24.4). PKC-β INHIBITION AND EXPERIMENTAL NEPHROPATHY The early stages of diabetic renal disease are characterized by glomerular hyper- filtration, mesangial expansion and microalbuminuria. Hyperglycaemia- induced de novo synthesis of DAG, coupled with activation of PKC, especially PKC-β, affects the structural and functional changes in the kidney via several dif- ferent mechanisms involving various phosphorylation substrates of PKC. For example, mesangial expansion has been attributed, in part, to PKC-mediated increases in transforming growth factor-β (TGFβ) gene expression, activation of cytosolic phospholipase A 2 and inhibition of Na + /K + -ATPase activity. 217 217 Fig. 24.4 Oral treatment with the PKC-β inhibitor ruboxistaurin, reverses diabetes- related reductions in Na + /K + -ATPase activity in retinal microvessels. Adapted from Diabetes 1998; 47: 464–469. Na + /K + -ATPase activity 40 35 30 25 20 15 10 5 0 Diabetes + ruboxistaurin * # DiabetesNormal CHAPTER 24 • EXPERIMENTAL PHARMACOLOGY USING ISOFORM-SELECTIVE PKC INHIBITORS SECTION IV • MECHANISMS OF HYPERGLYCAEMIA INDUCED VASCULAR DYSFUNCTION 218 Experimental studies with ruboxistaurin have shown that, following oral administration for eight weeks to STZ-diabetic and non-diabetic rats, urinary albumin excretion rate (AER) and glomerular hyperfiltration were signifi- cantly reduced (Fig. 24.3). Interestingly, higher doses of the PKC-β inhibitor (1–10 mg/kg/day) were required to inhibit diabetes-mediated PKC activation in the kidney compared with the retina (0.1 mg/kg/day). In addition, treat- ment with ruboxistaurin had no significant effect on GFR and filtration frac- tion in non-diabetic animals (Fig. 24.3). Among diabetic rats, however, the dose-response curve for ruboxistaurin in normalizing GFR paralleled its inhibitory effect on PKC activity. Renal protection with aminoguanidine and angiotensin- converting enzyme inhibition (ACE-I) involves normalization of glomerular PKC activity In experimental models of diabetic renal disease, e.g. the STZ-diabetic rat, it is well established that ACE-Is and aminoguanidine retard the structural and functional abnormalities characteristic of diabetic nephropathy, particularly with respect to reducing urinary AER. The exact mechanisms by which these therapeutic interventions work is not entirely clear, but recent work by George Jerums and colleagues has shown that glomerular PKC activity levels are normalized in STZ-diabetic rats during experimental treatment with aminoguanidine and the ACE-I, ramipril. Thus, diabetes-related increases in glomerular PKC activity may serve as an important common pathway by which metabolic and haemodynamic factors contribute to the initiation and progression of diabetic renal disease. Existing renoprotective agents, e.g. ACE-Is, may slow the progression of nephropathy, in part, by normalizing diabetes-induced increases in glomerular PKC activity. EFFECTS OF RUBOXISTAURIN IN EXPERIMENTAL DIABETIC NEUROPATHY Various pathways have been implicated in the pathogenesis of diabetic neu- ropathy, including increased polyol pathway activity, enhanced non-enzy- matic glycation and PKC activation. In addition, neural ischaemia is thought to play an important role in diabetic nerve injury, in part via PKC activation which impairs vasodilation and increases vasoconstrictor pathways in the endoneurial microvasculature. In experimental STZ-diabetic rats, motor nerve conduction velocity and sciatic nerve blood flow are reduced. Treatment with ruboxistaurin amelio- rated these abnormalities via mechanisms attributable to prevention of neu- ral ischaemia (Fig. 24.5). CHAPTER 24 • EXPERIMENTAL PHARMACOLOGY USING ISOFORM-SELECTIVE PKC INHIBITORS CLINICAL IMPLICATIONS OF AN ORALLY ACTIVE PKC-β INHIBITOR, RUBOXISTAURIN Extensive experimental studies have shown that ruboxistaurin selectively inhibits PKC-β in retinal, neural, renal and vascular tissues following oral administration without any significant adverse effects. The encouraging tol- erability profile of ruboxistaurin is no doubt attributable to its pharmaco- logical specificity for PKC-β I and PKC-β II. The animal studies have convinc- ingly shown that, following chronic oral treatment, ruboxistaurin amelio- rates the early increases in retinal blood flow, glomerular filtration rate and renal and retinal permeability. This data opens the possibility of a new and exciting pathway for therapeu- tic intervention in the earliest stages of diabetic microvascular disease. In par- ticular, such an approach would be unique in offering protection against the development and progression of retinopathy and nephropathy via a mecha- nism that is independent of (and complementary to) glucose or blood pres- sure reduction. Thus, in clinical practice, PKC-β inhibition would be used as an adjunct to all existing therapies for the prevention of diabetic vascular com- plications. Large multicentre clinical trials are on-going not only in diabetic retinopathy and renal disease but also in patients with other diabetic compli- cations, e.g. erectile dysfunction and diabetic neuropathy. 219 Fig. 24.5 Ruboxistaurin improves sciatic nerve conduction velocity in experimental models of peripheral neuropathy. A dose-related effect is illustrated. Velocity (m/s) Dose (mg/kg) 60 65 control level 55 0.0 0.1 0.3 1.0 10.0 25.0 SECTION IV • MECHANISMS OF HYPERGLYCAEMIA INDUCED VASCULAR DYSFUNCTION 220 FURTHER READING Aiello LP, Bursell SE, Clermont A et al. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective β-isoform-selective inhibitor. Diabetes 1997; 46: 1473–1480. Ishii H, Jirousek MR, Koya D et al. Amelioration of vascular dysfunction in diabetic rats by an oral PKC-β inhibitor. Science 1996; 272: 728–731. Kowluru RA, Jirousek MR, Stramm L et al. Abnormalities of retinal metabolism in diabetes or experimental galactosemia: V relationship between protein kinase C and ATPase. Diabetes 1998; 47: 464–469. Nakamura J, Kato K, Hamada Y et al. A protein kinase C-β-selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes 1999; 48: 2090–2095. CURRENT ISSUES • Ruboxistaurin is a unique orally active PKC inhibitor which is highly specific for the PKC-β isoforms. Following oral administration to STZ- diabetic rats, ruboxistaurin prevented diabetes-related increases in retinal and renal PKC activity in parallel with amelioration of glomerular hyperfiltration, microalbuminuria and increased retinal blood flow. • Ruboxistaurin shows an excellent tolerability profile in experimental diabetic animals, no doubt reflecting its specificity for inhibiting only two out of twelve PKC isoforms. Furthermore, in non-diabetic animals (in which there is no augmentation of PKC activity) ruboxistaurin has no significant effects on retinal or renal haemodynamics. Thus, the compound seems to be highly specific for PKC-β and only achieves therapeutic effects in experimental studies in which diabetes-related increases in PKC are present. • Large multicentre clinical trials with ruboxistaurin are on-going to assess its efficacy and safety in patients with diabetic retinopathy and peripheral neuropathy. In due course further studies will be established to define the role of this compound in other diabetes complications, including nephropathy and erectile dysfunction. INTRODUCTION Ruboxistaurin is the first molecule in an exciting new class of PKC-β‚ specific inhibitors which ameliorate the structural and functional vascular abnormali- ties associated with hyperglycaemia in humans and experimental animals. A series of detailed molecular and experimental studies were conducted to doc- ument the effects of ruboxistaurin in retinal, neural and endothelial tissues. These were followed by a series of multicentre clinical trials to evaluate longer term efficiency and safety in patients with diabetes related complications. The design and execution of these trials has posed considerable challenges, and many of these trials are still ongoing. There are, however, a number of encour- aging results already in the public domain from phase II studies. RUBOXISTAURIN IMPROVES ENDOTHELIAL DYSFUNCTION Diabetes is associated with endothelial dysfunction, and hyperglycaemia impairs the endothelial-dependent vasodilator response to acetylcholine. In a placebo controlled, double blind crossover study in healthy volunteers, there was evidence that ruboxistaurin improved forearm blood flow in response to incremental arterial infusions of the endothelium-dependent vasodilator methacholine under hyperglycaemic conditions (Fig. 25.1). Thus, this novel experimental study has confirmed that inhibition of PKC-β in healthy vol- unteers prevents the reduction in endothelium-dependent (nitric oxide mediated) vasodilation induced by acute hyperglycaemia. CLINICAL TRIALS OF RUBOXISTAURIN IN DIABETIC RETINOPATHY Experimental studies have shown that ruboxistaurin inhibits hypergly- caemia-induced PKC activation in the retina (Fig. 25.2). In addition ruboxis- taurin prevents neovascularization in a porcine model of retinal vein occlu- sion (Fig. 25.3). These experimental data provide encouraging evidence that PKC-β‚ inhibition might have a favourable effect on macular oedema forma- tion and new vessel formation (two sight threatening complications) in patients with diabetic retinopathy. The clinical development of ruboxistaurin began with phase I tolerability and pharmacokinetic studies in healthy volunteers, followed by phase II effi- cacy studies in patients with diabetes. In patients with type 1 or type 2 diabetes and minimal or no evidence of diabetic retinopathy, ruboxistaurin increased retinal blood flow in a dose-dependent manner, maximal after 32 mg daily for 221 CHAPTER 25 CLINICAL TRIALS WITH RUBOXISTAURIN Richard Donnelly MD, PhD, FRCP, FRACP Vascular Complications of Diabetes: Current Issues in Pathogenesis and Treatment, Second Edition Edited by Richard Donnelly, Edward Horton Copyright © 2005 by Blackwell Publishing Ltd SECTION IV • MECHANISMS OF HYPERGLYCAEMIA INDUCED VASCULAR DYSFUNCTION 222 Fig. 25.1 Forearm blood flow in healthy volunteers during euglycaemia and hyperglycaemia after pretreatment with ruboxistaurin or placebo. The PKC-β‚ inhibitor improved the endothelial-dependent vasodilator response to methacholine under conditions of high glucose. Adapted from Beckman et al. Circulation Research 2002; 90: 107–111. Forearm blood flow (ml / dl / min) 2 3 1 0 Placebo Ruboxistaurin p = 0.08 p = 0.001 Euglycaemia Hyperglycaemia Fig. 25.2 Ruboxistaurin attenuates the increase in retinal PKC activity in experimental rats with diabetes. PKC activity (pmo/min/mg of protein) 20 15 10 5 0 0 0.1 Ruboxistaurin (mg/kg/d) 10.0 Nondiabetic Diabetic CHAPTER 25 • CLINICAL TRIALS WITH RUBOXISTAURIN 223 Fig. 25.3 Ruboxistaurin prevents neovascularization in a porcine retinal vein occlusion model of new vessel formation. Neovascularization score 3 4 2 1 0 Placebo p = 0.03 Ruboxistaurin 1 mg/kg/d, po Fig. 25.4 Phase II study of ruboxistaurin in patients with type 1 or type 2 diabetes and retinopathy. In a double blind, placebo controlled study for four weeks, ruboxistaurin decreased mean retinal circulation time, i.e. improved retinal blood flow. Adapted from Aiello et al. Diabetes 1999; 48: A19. Extent of MCT abnormality at endpoint 1.0 1.2 0.4 0.2 0.6 0.8 0.0 Placebo 16 mg/d 32 mg/d [...]... 132 Diabetes in Early Pregnancy Study 136 diabetic amyotrophy 82, 83 227 228 INDEX Diabetic Control and Complications Trial (DCCT) 113 diabetic foot 105 10 amputation 105 , 106 , 107 , 125 angioplasty 107 callus formation 105 causes 105 –6, 105 education 107 healing 107 , 108 hyperbaric oxygen 107 management guidelines 125 PEDIS classification 106 prevention 107 recombinant platelet derived growth factor 108 ... 43 vascular risk assessment 38–9 see also myocardial infarction cost of diabetes 7 10, 9, 10 cotton wool spots 139, 141, 142, 143, 173, 174 cranial nerve palsy 174 cranial neuropathy 82 creatinine 26, 27, 31, 32, 73 cross-link formation 185 CT-angiography 26 cyclic GMP 53 cystoid macular oedema 157, 172 D DECODE study 182 delquamine 57 3-deoxyglucosone 179, 182 desipramine 116 Diabetes Control and Complications. .. stress 182–3, 184 carboxymethyl-lysine 179 cardiomyopathy 37, 193, 206 carotid stenoses 46 carpal tunnel syndrome 80 cataracts 171–2, 188 treatment 172 central retinal vein occlusion 173 cerebral haemorrhage 43, 45, 48 cerebral infarcts 44 cerebral small-artery disease 46 cerebrovascular disease see stroke Charcot neuroarthropathy 108 10, 108 , 109 management 110 X-rays 109 Charcot neuropathy 79 cholesterol... versus beta-blockers 63 fluorescein 102 , 153, 154, 155, 156, 174 acetyl-L-carnitine 124 angioplasty 40, 42, 44, 62, 66 acetylcholine 207, 221 diabetic foot 107 acetylcysteine 26 angiotensin receptor antagonists 30, 70 actin 199, 200 angiotensin-converting enzyme inhibitors see acupuncture 118 ACE inhibitors acute sensory neuropathy 80 ankle reflex 79, 80, 81, 122, 125 adenosine 53, 100 , 101 anti-arrhythmics... EURODIAB Controlled Trial of Lisinopril in Insulin-Dependent Diabetes Mellitus (EUCLID) 75, 93, 134 EURODIAB IDDM Complications Study 93 European Stroke Prevention Study 48, 70 external ocular muscle palsies 174 extracellular signal-regulated kinases 86 exudates 141, 142 exudative maculopathy 151 F F-actin 199 F-waves 101 ferritin 32 fibrates 43 fibrinogen 37, 62, 209 fidarestat 124 flame-shaped haemorrhages... microvascular disease 135 nephropathy 19, 23 retinopathy 135–6 type 1 diabetes 4 type 2 diabetes 4 GF109203X 213 GISSI-3 41, 63 glaucoma 146, 172 ghost-cell 166 haemolytic 166 neovascular 146, 172 glitazones 43 glomerular filtration rate 22, 31 glomerulopathy 209 10 glomerulosclerosis 25, 26, 185 GLUT-1 transporters 187, 189 glycaemic control, and cardiovascular risk 41–2, 42 glycine antagonists 49 glycoprotein... excretion rate 197 US Diabetes Control and Complications Trial (DCCT) 132 UTDWCS classification 106 uveitis 172, 174 V VA Cooperative Study on type 2 Diabetes Mellitus (VACSDM) 85 vardenafil 54 vascular adhesion molecule-1 209 vascular endothelial growth factor 89, 115 vascular permeability see endothelial permeability vascular permeability factor 166, 200, 202, 213 vascular risk assessment 38–9 vasodilators... insulin-like growth factor 89, 115 cystoid 157, 172 intercellular adhesion molecule-1 (ICAM-1) 209 duration of diabetes 152, 153 International Working Group of the Diabetic and retinopathy 152 Foot 106 maculopathy 139, 141, 151–61 intracavernosal injection therapy 56 diagnosis 153–6 intraocular pressure 137, 169 diffuse oedema 157 intraretinal haemorrhage 141 epidemiology 151 intraretinal microvascular... with mild to moderate non-proliferative diabetic retinopathy and non-visually threatening diabetic macular oedema will delay development of clinically-significant macular oedema or the need for laser photocoagulation The clinical trials with ruboxistaurin use 7-field stereoscopic fundal photographs and measurements of visual acuity as markers of drug efficacy CLINICAL TRIALS OF RUBOXISTAURIN IN DIABETIC... number of large phase III clinical trials are in progress to evaluate the effects of ruboxistaurin on various unpleasant symptoms of diabetic neuropathy and longer term outcomes in relation to nerve function and neurophysiological endpoints These trials use a combination of symptom scores and nerve function measurements to assess efficacy CLINICAL TRIALS OF RUBOXISTAURIN IN OTHER DIABETESRELATED COMPLICATIONS . Staurosporine PKC-α 360 45 PKC-β 1 4.7 23 PKC-β 2 5.9 19 PKC-γ 300 110 PKC-δ 250 28 PKC-ε 600 18 PKC-ζ > ;10 5 >1.5 × 10 3 PKC-η 52 5 Cyclic AMP kinase > ;10 5 100 Ca 2+ -calmodulin kinase 8 × 10 3 4 Casein. and Complications Trial (DCCT) 113 diabetic foot 105 10 amputation 105 , 106 , 107 , 125 angioplasty 107 callus formation 105 causes 105 –6, 105 education 107 healing 107 , 108 hyperbaric oxygen 107 management. 48 cerebral infarcts 44 cerebral small-artery disease 46 cerebrovascular disease see stroke Charcot neuroarthropathy 108 10, 108 , 109 management 110 X-rays 109 Charcot neuropathy 79 cholesterol and