**2. Pharmacological management of DPN via targeting pathogenetic mechanisms: From animal models to clinical practice**

#### **2.1. Managing metabolic derangements**

#### *2.1.1. Polyol pathway and aldose reductase inhibitors*

Polyol pathway arose as a plausible link of glucose dismetabolism to DPN in middle 1960s [43] and has received much interest due to the strong evidence accumulating from experimental diabetic rats [44]. Two consecutive oxidoreductive reactions essentially constitute the polyol pathway: the rate-limiting NADPH-dependent aldose reductase (AR) reduces glucose to sorbitol, which then becomes the substrate for NAD+ -dependent sorbitol dehydrogenase (SDH) and oxidized into fructose. Although AR has a high KM for glucose under the physio‐ logical condition, hyperglysolia (high intracellular glucose concentration) can excessively activate this enzyme resulting in a nearly 4-fold induction in glucose disposal through this pathway in human erythrocytes [45, 46]. Because these polyhydroxylated alcohols have little transmembrane diffusibility, their retention within ocular lens fibrils of hyperglycemic rats or rabbits was proposed to cause hyperosmotic perturbation of intracellular metabolites, electrolytes and other osmolytes and subsequent hydropic cataractogenesis as observed. All but highly disabling late sequelae of DPN include limb ischemia and joint deformity [6]; the latter also being termed Charcot's neuroarthropathy or Charcot's joints [1]. In addition to significant morbidities, several separate cohort studies provided evidence that DPN [29], diabetic foot ulcers [30] and increased toe vibration perception threshold (VPT) [31] are all independent risk factors for mortality. Overall, neuropathic pain, foot complication as well as various associated psychosocial comorbidities inflict a significant diminution on the quality and duration of life of individuals affected by DPN, which in turn is raising an escalating

Unfortunately, current therapy for DPN is far from effective and at best only delays the onset and/or progression of the disease via tight glucose control, the only established means for managing diabetic complications in the U.S. Several large-scale, multicenter and landmark clinical studies, including Diabetes Control and Complication Trial, provided irrefutable evidence that chronic hyperglycemia is a leading factor in the etiology and treatment of DPN [32-36]. However, euglycemia cannot always be achieved through aggressive insulin therapy or other anti-diabetic agents. Even with near normoglycemic control, a substantial proportion of patients still suffer the debilitating neurotoxic consequences of diabetes [34]. On the other hand, some with poor glucose control are spared from clinically evident signs and symptoms of neuropathy for a long time after diagnosis [37-39]. Thus, other etiological factors independent of hyperglycemia are likely to be involved in the development of DPN. Data from a number of prospective, observational studies suggested that older age, longer diabetes duration, genetic polymorphism, presence of cardiovascular disease markers, malnutrition, presence of other microvascular complications, alcohol and tobacco consumption, and higher constitutional indexes (e.g. weight and height) interact with diabetes and make for strong predictors of neurological decline [13, 32, 40-42].

Meanwhile, enormous efforts have been devoted to understanding and intervening with the molecular and biochemical processes linking the metabolic disturbances to sensorimotor deficits by studying diabetic animal models. In return, nearly 2,200 articles were published in PubMed central and at least 100 clinical trials were reported evaluating the efficacy of a number of pharmacological agents; the majority of them are designed to inhibit specific pathogenic mechanisms identified by these experimental approaches. Candidate agents have included aldose reductase inhibitors, AGE inhibitors, γ-linolenic acid, α-lipoic acid, vasodilators, nerve growth factor, protein kinase Cβ inhibitors, and vascular endothelial growth factor. Notwithstanding a fruitful of knowledge and promising results in animals, none has translated into definitive clinical success (Figure 1). While the notorious biochemical heterogeneity and temporal non-uniformity of the disease processes among and even within individuals can take much of the blame, investigators must take into serious consideration the marked differences between animals and humans, which may substantially impair the application of experimental data to clinical settings. The following sections of this chapter describe the clinical outcomes of these pathogenetic treatments that put previous observations generated by animal studies into perspective,

Figure 1. Summary of Current Clinical Status of Anti-DPN Drugs Developed via Animal Models. Data are generated from published experimental and clinical results to date on pharmacological agents (a total of 23 drugs) targeting pathogenetic mechanisms listed in but not limited to section 2.

**Withdrawn (Toxicity)**

**Efficacy)**

**Status Pending**

**Withdrawn (Lack of Efficacy) Withdrawn (Toxicity & Lack of**

**Approved with Marginal Benefits**


Targeting some of these modifiable risk factors in addition to glycemia may improve the management of DPN.

and discuss the molecular, cellular and physiological roots underlying the limited translation.

32 Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder

**Clinical Status of Anti-DPN Drugs Identified Through Animal Models**

**17%**

**44%**

**Figure 1.** Summary of Current Clinical Status of Anti-DPN Drugs Developed via Animal Models. Data are generated from published experimental and clinical results to date on pharmacological agents (a total of 23 drugs) targeting

Polyol pathway arose as a plausible link of glucose dismetabolism to DPN in middle 1960s [43] and has received much interest due to the strong evidence accumulating from experimental diabetic rats [44]. Two consecutive oxidoreductive reactions essentially constitute the polyol pathway: the rate-limiting NADPH-dependent aldose reductase (AR) reduces glucose to

(SDH) and oxidized into fructose. Although AR has a high KM for glucose under the physio‐ logical condition, hyperglysolia (high intracellular glucose concentration) can excessively activate this enzyme resulting in a nearly 4-fold induction in glucose disposal through this pathway in human erythrocytes [45, 46]. Because these polyhydroxylated alcohols have little transmembrane diffusibility, their retention within ocular lens fibrils of hyperglycemic rats or rabbits was proposed to cause hyperosmotic perturbation of intracellular metabolites, electrolytes and other osmolytes and subsequent hydropic cataractogenesis as observed. All

**2. Pharmacological management of DPN via targeting pathogenetic**

**mechanisms: From animal models to clinical practice**

**17%**

pathogenetic mechanisms listed in but not limited to section 2.

**2.1. Managing metabolic derangements**

*2.1.1. Polyol pathway and aldose reductase inhibitors*

sorbitol, which then becomes the substrate for NAD+

**13%**

**9%**

health, social and economic problem in both developed and developing countries [4, 14].

**1.2. A medical challenge** 

of these were preventable and reversible by blocking AR [47-51]. In mice, transgenic overex‐ pression of the gene encoding human AR (hAR) in lens epithelia submitted these cataractresistant animals to sugar-induced polyol deposit and cataract formation, which became more acute when coupled with genetic SDH deficiency [52]. Studies of type 1 and type 2 diabetes models, including alloxan- and streptozotocin (STZ)-induced diabetic rats and leptin-deficient *ob*/*ob* mice, soon confirmed a significant elevation of sorbitol and fructose in sciatic nerves, dorsal root ganglia (DRGs) and spinal cord. This correlated with nerve/axonal conduction and transport deficiencies, loss of intraepidermal nerve fibers, increased neural and endoneurial oxidative-nitrosative stress as well as thermal hypoalgesia and tactile allodynia [43, 53-57]. A "polyol hypothesis" derived from diabetic lens was thus propelled to the pathogenesis of DPN [47]. In keeping with this notion, AR inhibitors that reduce nerve polyol levels showed remarkable preservation of nerve structure and function in rats with either spontaneous or chemical-induced diabetes [53, 58-60]. Systemic hAR overexpression combined with STZinduced diabetes led to an exacerbated but AR inhibitor-preventable peripheral nerve sorbitol and fructose buildup, electroactivity suppression and myelinated fiber atrophy [61]. A similar biochemical and electrophysiological but not morphological abnormality was obtained with Schwann cell (SC)-targeted hAR transgenic mice, indicating that SC AR hyperactivity con‐ tributes to many, though not all pathological change of DPN [62]. Conversely, AR-knockout mice showed no obvious sorbitol accumulation, conduction slowing, oxidative stress, or stress kinase activation. Additionally, there were fewer loss of sural nerve fibers in AR-deficient mice compared to wild-types (WTs) [63]. Since galactose has approximately 4 times higher affinity for AR than glucose [64] and its reduction product galactitol is poorly disposed, galactose-rich diet was used as a popular substitute for classical hyperglycemic models to exemplify and examine the role of excessive polyalcohol formation in the genesis of diabetic cataract and neuropathy [47]. Along the line with "aldo-osmotic theory", galactosemic rodents that accrue much greater level of this alternate AR metabolite also exhibit similar and sometimes more severe electrophysiological, anatomical and biochemical defects that are seen diabetic models [65-67]. However, galactosemia is a rare metabolic condition in humans (less than 0.002% of the population) [68] and the galactosemic lens and nerves often manifest functional and structural lesions resulting from acute and exaggerated galactitol intoxication that differ from those of diabetic cataract and neuropathy [47, 69-71]. Hence, galactose-fed animals are neither appropriate models for studying diabetic complications nor good replacements for character‐ izing the pathogenetic involvement of sorbitol pathway in these conditions. Other studies further revealed that neither the morphometrical [59] nor functional indices in DPN correlate with the tissue sorbitol content [72, 73]. Instead, nerve myo-inositol content is more closely related to the neurophysiological function according to most reports. Depletion of cytoplasmic myo-inositol, protein kinase C activation and tubulin/Na+ /K+ -ATPase complex formation were proposed mechanisms that mediate polyol pathway overflow-induced impairment of Na+ /K + -ATPase ion pumping and subsequent reduction of nerve conduction velocity (NCV) [45, 55, 74]. In addition, augmented cofactor consumption by AR and SDH not only deprives gluta‐ thione reductase of NADPH and the capacity to regenerate reduced glutathione (GSH) [45] but also contributes to an imbalanced redox state of NADH/NAD+ [75], thus promoting oxidative and vascular injury [63, 76, 77].

without causing serious adverse reactions [111, 112]. Ranirestat, or As-3201, emerged as a spirosuccinimide with a better drug profile, and was effective in increasing NCVs and sensory function in a phase II trial of mild to moderate diabetic sensorimotor polyneuropathy [113]. The large-scale long-term Phase III trial of Ranirestat, however, did not show statistically significant differences in sensory parameters compared to placebo at all doses tested [114]. Another spirohydantoin, Fidarestat, displayed increased tolerability and a similar degree of improvement in subjective measures to that of Sorbinil [115]. After phase III evaluation, a minor therapeutic value was concluded for Fidarestat in the literatures and its further development was suspended for financial reasons [14, 116]. To date, Epalrestat is the only ARI approved for clinical use in Japan. Despite its success in delaying nerve conduction and sensory abnormalities in a randomized, open label, controlled multicenter trial among Japanese patients [117], the efficacy of Epalrestat has not been confirmed in other populations and appears only marginal in other documentations [1, 118]. In an attempt to identify a meaningful treatment effect of ARIs for clinical DPN, Chalk *et al* conducted a meta-analysis for 13 trials of ARIs involving 879 treated participants and 909 controls. This report found no difference in the overall outcome (SMD -0.25, 95% CI -0.56 to 0.05), nerve conduction parameters or foot ulcers between treatment and control group [119]. Similarly, a previous meta-analysis of studies published before 1996 testing four different ARIs indicated that AR inhibition achieved less than 1 m/s offsets in the decline of median and peroneal motor nerve conduction velocity (MNCV) as the single true statistical change [120]. Given these inconclusive results and safety issues, FDA has not approved any of the aforementioned agents for pharmacological inter‐ vention of DPN. Although a number of confounding factors, including unexpected placebo effect and trial design, have been blamed for the disappointing clinical outcome, the lack of clear sensory protection by ARIs puts the relevance of polyol pathway to DPN into question.

From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of…

http://dx.doi.org/10.5772/55364

35

Animal and cell studies have well established the contribution of advanced glycation end products (AGEs) to diabetic tissue damage. Nerves, retina and kidney do not depend on insulin for glucose uptake and absorb this energy substrate as a direct function of the circulating glucose concentration. Prolonged hyperglysolia cultivates the glucose autoxidation, decom‐ position of the Amadori products (fructosamines) following adduction of glucose to the amino groups of lysine residues in the proteins, and fragmentation of glycolytic intermediates (such as glyceraldehyde-3-phosphate and dihydroxyacetone phosphate). All of these gives rise to glyoxal, 3-deoxyglucosone and methylglyoxal within the cells [121]. These highly reducing dicarbonyls are AGE precursors or glycating agents that non-enzymatically react with intracellular nucleotides, proteins, lipids, extracellular matrix and plasma components [122]. The last one is best reflected by the elevated serum glycosylated hemoglobin [HbA1c] level in diabetes. AGE modification of growth factors [123], endocytotic proteins [124], cytoskeletal actin and filaments [125, 126], interstitial matrix and adhesive molecules [127] as well as serum albumin [128] were found in increased amounts in hyperglycemia-treated endothelial cells or diabetic rats and these associated with increased vascular damage, endocytosis, cytoskeletal disassembly, fluid filtration and albuminuria. In both human diabetics and STZ-rats, there was enhanced AGE deposition in peripheral nerves compared to healthy controls as indicated by

*2.1.2. Advanced glycation and aminoguanidine*

Overall, the above and numerous other observations obtained from the use of animal models demonstrated consistently that increased polyol metabolism is a strong and readily reversible component in the pathogenesis of diabetes-induced degenerative changes. However, data from human studies indicated no convincing association between the elevation of glucose flux via AR and neuropathic development. Whereas nerves from amputated limbs of diabetic individuals contained significantly higher concentrations of sorbitol and fructose than nondiabetics [78], an assessment of sural nerve biopsies by Dyck *et al.* found that over two thirds of subjects with mild to severe clinical signs or symptoms of DPN had a normal polyol content [79]. A later study by the same group was able to show an inverse relationship between nerve sorbitol level and myelinated fiber density but not other neurological parameters [80]. Importantly, none of the nerve specimen analyses identified a decrease in myo-inositols in relation to DPN, in contrast with the invariable observations of myo-inositol deficiency in rodent models. Likewise, dietary supplementation of myo-inositol prevented and reversed a variety of pathophysiological processes associated with early DPN in rats [81, 82] but failed to normalize any peripheral nerve deterioration in patients with a recent diabetes onset [83, 84]. Nevertheless, the prominent success of AR inhibitors (ARIs) in preventing and reversing experimental diabetic cataract and neuropathy [58, 60, 85-89] as well as the findings of AR gene polymorphisms in diabetic microvascular complication [90-93] spurred a broad enthusiasm in the clinical exploration of these ARIs. While the use of various ARIs almost always prevented or reversed the lens opacification in diabetic rats [94], whether they can reduce the risk of cataract formation in human diabetics remains unclear. This is because most experimentally induced diabetic cataracts occur acutely and possess distinct morphological alterations similar to the features seen with the rare juvenile form of diabetic cataract. Contrasting the juvenile form, the majority of cataracts in diabetes has a dubious sorbitol increase and is represented by the slow, refractive cataract change in diabetic adults [95]. Therefore, a direct evaluation of the use of ARIs as an anti-cataract treatment is difficult in these animal models.

With regard to DPN, two earliest ARIs to be tested for their clinical efficacy in treating DPN were Alrestatin and Sorbinil, which were the prototypic ARIs belonging to the chemical classes of succinimide and spirohydantoins, respectively. Alrestatin produced minor subjective benefit but no improvement on NCV or other objective examinations [96, 97]. While Sorbinil moderately reduced the NCV decline and increased the density of regenerating myelinated fibers in sural nerves [98, 99], its influence on pain and vagal function is questionable and no meaningful therapeutic effects were experienced by patients with diabetic autonomic or polyneuropathy [100-102]. Both Sorbinil and Alrestatin were withdrawn from the clinical setting due to a high rate of toxicity involving photosensitive skin rash [1, 14]. Tolrestat, an acetic acid compound, was able to halt the progression of subclinical peripheral and autonomic deficits in a 52-week duration but had only a mild benefit on chronic symptomatic sensori‐ motor neuropathy [103-106]. The poor electrophysiological outcome and the incidence of fatal hepatic necrosis eventually led to discontinuation of Tolrestat study [107]. In the cases of the carboxylic acid class of ARIs, Ponalrestat manifested minimal tissue penetration and nerve sorbitol reduction, in spite of its good pharmacokinetics and pharmacodynamics in diabetic rats [108-110]. Although Zopolrestat and Zenarestat demonstrated a dose-dependent amelio‐ ration in NCV deficits, both of them failed to significantly improve the clinical endpoints without causing serious adverse reactions [111, 112]. Ranirestat, or As-3201, emerged as a spirosuccinimide with a better drug profile, and was effective in increasing NCVs and sensory function in a phase II trial of mild to moderate diabetic sensorimotor polyneuropathy [113]. The large-scale long-term Phase III trial of Ranirestat, however, did not show statistically significant differences in sensory parameters compared to placebo at all doses tested [114]. Another spirohydantoin, Fidarestat, displayed increased tolerability and a similar degree of improvement in subjective measures to that of Sorbinil [115]. After phase III evaluation, a minor therapeutic value was concluded for Fidarestat in the literatures and its further development was suspended for financial reasons [14, 116]. To date, Epalrestat is the only ARI approved for clinical use in Japan. Despite its success in delaying nerve conduction and sensory abnormalities in a randomized, open label, controlled multicenter trial among Japanese patients [117], the efficacy of Epalrestat has not been confirmed in other populations and appears only marginal in other documentations [1, 118]. In an attempt to identify a meaningful treatment effect of ARIs for clinical DPN, Chalk *et al* conducted a meta-analysis for 13 trials of ARIs involving 879 treated participants and 909 controls. This report found no difference in the overall outcome (SMD -0.25, 95% CI -0.56 to 0.05), nerve conduction parameters or foot ulcers between treatment and control group [119]. Similarly, a previous meta-analysis of studies published before 1996 testing four different ARIs indicated that AR inhibition achieved less than 1 m/s offsets in the decline of median and peroneal motor nerve conduction velocity (MNCV) as the single true statistical change [120]. Given these inconclusive results and safety issues, FDA has not approved any of the aforementioned agents for pharmacological inter‐ vention of DPN. Although a number of confounding factors, including unexpected placebo effect and trial design, have been blamed for the disappointing clinical outcome, the lack of clear sensory protection by ARIs puts the relevance of polyol pathway to DPN into question.

#### *2.1.2. Advanced glycation and aminoguanidine*

Overall, the above and numerous other observations obtained from the use of animal models demonstrated consistently that increased polyol metabolism is a strong and readily reversible component in the pathogenesis of diabetes-induced degenerative changes. However, data from human studies indicated no convincing association between the elevation of glucose flux via AR and neuropathic development. Whereas nerves from amputated limbs of diabetic individuals contained significantly higher concentrations of sorbitol and fructose than nondiabetics [78], an assessment of sural nerve biopsies by Dyck *et al.* found that over two thirds of subjects with mild to severe clinical signs or symptoms of DPN had a normal polyol content [79]. A later study by the same group was able to show an inverse relationship between nerve sorbitol level and myelinated fiber density but not other neurological parameters [80]. Importantly, none of the nerve specimen analyses identified a decrease in myo-inositols in relation to DPN, in contrast with the invariable observations of myo-inositol deficiency in rodent models. Likewise, dietary supplementation of myo-inositol prevented and reversed a variety of pathophysiological processes associated with early DPN in rats [81, 82] but failed to normalize any peripheral nerve deterioration in patients with a recent diabetes onset [83, 84]. Nevertheless, the prominent success of AR inhibitors (ARIs) in preventing and reversing experimental diabetic cataract and neuropathy [58, 60, 85-89] as well as the findings of AR gene polymorphisms in diabetic microvascular complication [90-93] spurred a broad enthusiasm in the clinical exploration of these ARIs. While the use of various ARIs almost always prevented or reversed the lens opacification in diabetic rats [94], whether they can reduce the risk of cataract formation in human diabetics remains unclear. This is because most experimentally induced diabetic cataracts occur acutely and possess distinct morphological alterations similar to the features seen with the rare juvenile form of diabetic cataract. Contrasting the juvenile form, the majority of cataracts in diabetes has a dubious sorbitol increase and is represented by the slow, refractive cataract change in diabetic adults [95]. Therefore, a direct evaluation of

34 Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder

the use of ARIs as an anti-cataract treatment is difficult in these animal models.

With regard to DPN, two earliest ARIs to be tested for their clinical efficacy in treating DPN were Alrestatin and Sorbinil, which were the prototypic ARIs belonging to the chemical classes of succinimide and spirohydantoins, respectively. Alrestatin produced minor subjective benefit but no improvement on NCV or other objective examinations [96, 97]. While Sorbinil moderately reduced the NCV decline and increased the density of regenerating myelinated fibers in sural nerves [98, 99], its influence on pain and vagal function is questionable and no meaningful therapeutic effects were experienced by patients with diabetic autonomic or polyneuropathy [100-102]. Both Sorbinil and Alrestatin were withdrawn from the clinical setting due to a high rate of toxicity involving photosensitive skin rash [1, 14]. Tolrestat, an acetic acid compound, was able to halt the progression of subclinical peripheral and autonomic deficits in a 52-week duration but had only a mild benefit on chronic symptomatic sensori‐ motor neuropathy [103-106]. The poor electrophysiological outcome and the incidence of fatal hepatic necrosis eventually led to discontinuation of Tolrestat study [107]. In the cases of the carboxylic acid class of ARIs, Ponalrestat manifested minimal tissue penetration and nerve sorbitol reduction, in spite of its good pharmacokinetics and pharmacodynamics in diabetic rats [108-110]. Although Zopolrestat and Zenarestat demonstrated a dose-dependent amelio‐ ration in NCV deficits, both of them failed to significantly improve the clinical endpoints

Animal and cell studies have well established the contribution of advanced glycation end products (AGEs) to diabetic tissue damage. Nerves, retina and kidney do not depend on insulin for glucose uptake and absorb this energy substrate as a direct function of the circulating glucose concentration. Prolonged hyperglysolia cultivates the glucose autoxidation, decom‐ position of the Amadori products (fructosamines) following adduction of glucose to the amino groups of lysine residues in the proteins, and fragmentation of glycolytic intermediates (such as glyceraldehyde-3-phosphate and dihydroxyacetone phosphate). All of these gives rise to glyoxal, 3-deoxyglucosone and methylglyoxal within the cells [121]. These highly reducing dicarbonyls are AGE precursors or glycating agents that non-enzymatically react with intracellular nucleotides, proteins, lipids, extracellular matrix and plasma components [122]. The last one is best reflected by the elevated serum glycosylated hemoglobin [HbA1c] level in diabetes. AGE modification of growth factors [123], endocytotic proteins [124], cytoskeletal actin and filaments [125, 126], interstitial matrix and adhesive molecules [127] as well as serum albumin [128] were found in increased amounts in hyperglycemia-treated endothelial cells or diabetic rats and these associated with increased vascular damage, endocytosis, cytoskeletal disassembly, fluid filtration and albuminuria. In both human diabetics and STZ-rats, there was enhanced AGE deposition in peripheral nerves compared to healthy controls as indicated by immunohistochemical assay [129, 130]. Particularly, pentosidine, a long-lived AGE marker, was significantly elevated in the cytoskeletal protein extracts isolated from diabetic subjects [130, 131]. Moreover, nerve specimens that harvest more AGEs also manifest lower myelinated fiber density.

PKC activation to diminished Na+

/K+

nism of Na+

application.

antagonists and agonists normalized Na+

**2.3. Increasing neurotrophic support**

*2.3.1. Growth factors and growth factor replacement therapy*

/K+

/K+

suggesting a conflicting involvement of PKC enhancement and diminishment in the mecha‐

selective inhibitor, LY333531, restored sciatic nerve blood flow and NCVs in STZ-induced diabetes [150, 151]. In addition, little data from humans, if any, has been obtained to support a PKC change in diabetic peripheral nerves. These experimental results nonetheless implicated PKC inhibition as a prospective avenue for anti-diabetic complication to investigators. The same inhibitor LY333531 (by Eli Lily) with a generic name Ruboxistaurin entered clinical evaluation as a treatment for DPN. In the trial of a small cohort of patients, Vinik *et al* reported that a 32 mg/day Ruboxistaurin for 6 months elicited significant alleviation on skin microvas‐ cular blood flow, total sensory symptoms and quality of life [152]. Recently, a 18-week treatment of Ruboxistaurin to a smaller subset of patients with type 2 diabetes proved beneficial in improving total symptom score (NTSS-6) and quality of life [153]. Unfortunately, this did not translate to a multinational, randomized, phase II, double-blind, placebo-control‐ led study consisting of 205 patients at an equal or double dosage of Ruboxistaurin [154]. Although Ruboxistaurin is well tolerated, Eli Lily withdrew its marketing authorization

Mammalian nervous system depends on a group of endogenous and heterogeneous biomo‐ lecules for proper physiological functions including growth, survival, differentiation and regeneration. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from the neurotrophin family are retrogradely transported to neuronal cell bodies after secretion from organs innervated by nerve terminals. These three neurotro‐ phins regulate the activity of small nociceptive and sympathetic sensory fibers, medium size sensory and motor fibers, large diameter sensorimotor and sympathetic neurons, respectively [155]. Other frequently studied growth factors in this context are glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) as well as insulin-like growth factor-1 (IGF-1), which are expressed by peripheral glia and/or neurons and manifest diverse trophic effects on sensory, motor and autonomic nerves [156]. In experimental rodent models, the protein and/or mRNA levels of NGF, BDNF and NT-3 have been observed to both upregulate and downregulate in peripheral nerves, sensory glia and such target tissues as skin keratino‐ cytes, skeletal muscles and submandibular glands [157-164]. Despite these conflicting reports, it is generally believed that the retrograde and anterograde axonal transport of these neuro‐ trophins are diminished in diabetic nerves [14, 165]. Similarly, IGF-1 and CNTF were found to be reduced in various tissues examined in type 1 and type 2 diabetic rat models [166-168]. In STZ or diabetic BB/Wor rats, deficient NGF and IGF-1 level correlated with inadequate macrophage recruitment and Wallerian degeneration after sciatic nerve injury [166, 169]. As postulated by the authors and others, this may explain the perturbed nerve regeneration in diabetes. Considering the highly dynamic nerve degeneration/regeneration in the initial stage


From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of…


pumping in peripheral nerves of diabetic animals,

http://dx.doi.org/10.5772/55364

37

With respect to intervention, aminoguanidine was the earliest chemical characterized for its anti-glycation activity. It is a hydrazine that preferably and competitively binds to AGE precursors and prevents further irreversible protein glycation [132]. Later studies discovered that besides inhibition of AGE formation, aminoguanidine can negatively act on inducible nitric oxide synthase [133], amine oxidase [134] and reactive oxygen species [135]. Such plethoric pharmacological properties suggest that aminoguanidine is not an appropriate investigational tool for the role of advance glycation in diabetic pathology. However, the continuous use of this compound in preclinical and clinical research was justified by its promising therapeutic effects in rat model of diabetic nephropathy [136], retinopathy [136] and neuropathy [137]. Whereas treating diabetic rats with various doses of aminoguanidine prevented or ameliorated the decrease in nerve blood flow, slowing of NCVs, endoneurial microvessel expansion and failure of sensory nerve regeneration [137-141], subcutaneous injection of aminoguanidine did not improve any of the structural or functional abnormalities in STZ-induced type I diabetic baboons [142]. Although the authors concluded that accumu‐ lation of AGEs is not likely an early mechanism of nerve damage in DPN, this discrepancy may also reflect considerable species differences. Indeed, none of the large standardized clinical trials proved a significant advantage of aminoguanidine over placebo in patients, who had well-established diabetic nephropathy [143, 144]. Rather, aminoguanidine adversely affected gastrointestinal, hepatic, respiratory and immune functions and finally led to termination of the studies. For these reasons, no further evaluation of the efficacy of amino‐ guanidine in treating DPN was pursued.

#### **2.2. Blocking signaling conducers**

#### *2.2.1. Protein kinase C and ruboxistaurin*

Protein kinase C (PKC) is a ubiquitous serine/threonine kinase of numerous isoforms and cellular functions. Observations in retinal and glomerular tissues from diabetic animals *in vitro* and *in vivo* support the hypothesis that elevated glycolysis subsequent to hyperglycemia dramatically raises 1,2-diacylglycerol (DAG) synthesis. In turn, DAG activates a majority of PKC family members, including PKC-α and -β [45]. Enhanced expression and activity of PKC isoforms, primarily PKC-β, pathologically affect vascular contractility and permeability thereby compromising microcirculation and causing microvascular occlusion [14, 145]. These deleterious consequences have been suggested by many to contribute to the vascular insults and development of retinopathy, nephropathy and cardiovascular disorder in diabetes. However, DAG and PKC upregulation is not a uniform pattern of change in every complica‐ tion-prone tissue. Unlike the findings in nonneural diabetic complications, nerve DAG levels fall in diabetes and experimental rodent models have presented decreased, increased and unaltered PKC activity [146-148]. Studies of mesangial and smooth muscle cells have linked PKC activation to diminished Na+ /K+ -ATPase function [149]. On the other hand, both PKC antagonists and agonists normalized Na+ /K+ pumping in peripheral nerves of diabetic animals, suggesting a conflicting involvement of PKC enhancement and diminishment in the mecha‐ nism of Na+ /K+ -ATPase deficits [146]. It is thus intriguing how administration of a PKC-β selective inhibitor, LY333531, restored sciatic nerve blood flow and NCVs in STZ-induced diabetes [150, 151]. In addition, little data from humans, if any, has been obtained to support a PKC change in diabetic peripheral nerves. These experimental results nonetheless implicated PKC inhibition as a prospective avenue for anti-diabetic complication to investigators. The same inhibitor LY333531 (by Eli Lily) with a generic name Ruboxistaurin entered clinical evaluation as a treatment for DPN. In the trial of a small cohort of patients, Vinik *et al* reported that a 32 mg/day Ruboxistaurin for 6 months elicited significant alleviation on skin microvas‐ cular blood flow, total sensory symptoms and quality of life [152]. Recently, a 18-week treatment of Ruboxistaurin to a smaller subset of patients with type 2 diabetes proved beneficial in improving total symptom score (NTSS-6) and quality of life [153]. Unfortunately, this did not translate to a multinational, randomized, phase II, double-blind, placebo-control‐ led study consisting of 205 patients at an equal or double dosage of Ruboxistaurin [154]. Although Ruboxistaurin is well tolerated, Eli Lily withdrew its marketing authorization application.

#### **2.3. Increasing neurotrophic support**

immunohistochemical assay [129, 130]. Particularly, pentosidine, a long-lived AGE marker, was significantly elevated in the cytoskeletal protein extracts isolated from diabetic subjects [130, 131]. Moreover, nerve specimens that harvest more AGEs also manifest lower myelinated

36 Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder

With respect to intervention, aminoguanidine was the earliest chemical characterized for its anti-glycation activity. It is a hydrazine that preferably and competitively binds to AGE precursors and prevents further irreversible protein glycation [132]. Later studies discovered that besides inhibition of AGE formation, aminoguanidine can negatively act on inducible nitric oxide synthase [133], amine oxidase [134] and reactive oxygen species [135]. Such plethoric pharmacological properties suggest that aminoguanidine is not an appropriate investigational tool for the role of advance glycation in diabetic pathology. However, the continuous use of this compound in preclinical and clinical research was justified by its promising therapeutic effects in rat model of diabetic nephropathy [136], retinopathy [136] and neuropathy [137]. Whereas treating diabetic rats with various doses of aminoguanidine prevented or ameliorated the decrease in nerve blood flow, slowing of NCVs, endoneurial microvessel expansion and failure of sensory nerve regeneration [137-141], subcutaneous injection of aminoguanidine did not improve any of the structural or functional abnormalities in STZ-induced type I diabetic baboons [142]. Although the authors concluded that accumu‐ lation of AGEs is not likely an early mechanism of nerve damage in DPN, this discrepancy may also reflect considerable species differences. Indeed, none of the large standardized clinical trials proved a significant advantage of aminoguanidine over placebo in patients, who had well-established diabetic nephropathy [143, 144]. Rather, aminoguanidine adversely affected gastrointestinal, hepatic, respiratory and immune functions and finally led to termination of the studies. For these reasons, no further evaluation of the efficacy of amino‐

Protein kinase C (PKC) is a ubiquitous serine/threonine kinase of numerous isoforms and cellular functions. Observations in retinal and glomerular tissues from diabetic animals *in vitro* and *in vivo* support the hypothesis that elevated glycolysis subsequent to hyperglycemia dramatically raises 1,2-diacylglycerol (DAG) synthesis. In turn, DAG activates a majority of PKC family members, including PKC-α and -β [45]. Enhanced expression and activity of PKC isoforms, primarily PKC-β, pathologically affect vascular contractility and permeability thereby compromising microcirculation and causing microvascular occlusion [14, 145]. These deleterious consequences have been suggested by many to contribute to the vascular insults and development of retinopathy, nephropathy and cardiovascular disorder in diabetes. However, DAG and PKC upregulation is not a uniform pattern of change in every complica‐ tion-prone tissue. Unlike the findings in nonneural diabetic complications, nerve DAG levels fall in diabetes and experimental rodent models have presented decreased, increased and unaltered PKC activity [146-148]. Studies of mesangial and smooth muscle cells have linked

fiber density.

guanidine in treating DPN was pursued.

**2.2. Blocking signaling conducers**

*2.2.1. Protein kinase C and ruboxistaurin*

#### *2.3.1. Growth factors and growth factor replacement therapy*

Mammalian nervous system depends on a group of endogenous and heterogeneous biomo‐ lecules for proper physiological functions including growth, survival, differentiation and regeneration. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from the neurotrophin family are retrogradely transported to neuronal cell bodies after secretion from organs innervated by nerve terminals. These three neurotro‐ phins regulate the activity of small nociceptive and sympathetic sensory fibers, medium size sensory and motor fibers, large diameter sensorimotor and sympathetic neurons, respectively [155]. Other frequently studied growth factors in this context are glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) as well as insulin-like growth factor-1 (IGF-1), which are expressed by peripheral glia and/or neurons and manifest diverse trophic effects on sensory, motor and autonomic nerves [156]. In experimental rodent models, the protein and/or mRNA levels of NGF, BDNF and NT-3 have been observed to both upregulate and downregulate in peripheral nerves, sensory glia and such target tissues as skin keratino‐ cytes, skeletal muscles and submandibular glands [157-164]. Despite these conflicting reports, it is generally believed that the retrograde and anterograde axonal transport of these neuro‐ trophins are diminished in diabetic nerves [14, 165]. Similarly, IGF-1 and CNTF were found to be reduced in various tissues examined in type 1 and type 2 diabetic rat models [166-168]. In STZ or diabetic BB/Wor rats, deficient NGF and IGF-1 level correlated with inadequate macrophage recruitment and Wallerian degeneration after sciatic nerve injury [166, 169]. As postulated by the authors and others, this may explain the perturbed nerve regeneration in diabetes. Considering the highly dynamic nerve degeneration/regeneration in the initial stage of DPN, growth factor therapy early in disease progression may minimize the damage and aid the axonal repair. To this end, abundant support has been produced using an array of spontaneous, chemical or transgenic diabetic models administered recombinant growth factors. Of note, NGF treatment restored neuropeptide level, C-fiber function and dermal myelinated innervation, alleviated neuropathic pain and promoted injury repair [156]. Whereas BDNF and NT-3 elicited a preferential attenuation in the structural and functional changes of large myelinated sensory and motor fibers [14], GDNF and IGF-1 showed a broad preservation of somatic and autonomic nervous system [19, 156]. Moreover, CNTF adminis‐ tration prevented/rescued behavioral and electrophysiological dysfunction, and enhanced sensory nerve resprouting in rats previously injected STZ [168, 170].

Other than NGF, a double-blind, placebo-controlled study was also conducted for rhBDNF but found no evidence of improvements on the primary endpoints associated with diabetic sensory neuropathy [190]. Although rhGDNF and rhNT-3 were supposed to enter early clinical assessments for DPN management, they have not yielded any clinical report except the withdrawal of NT-3 from phase I study [156]. It is therefore apparent that the expected outcomes were not met. For IGF-1 and CNTF, development of replacement therapy is also hindered by their non-specific impacts on the central nervous system [191] and muscles [192],

From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of…

http://dx.doi.org/10.5772/55364

39

Multiple epidemiological analyses have previously identified that hypertension strongly increases the occurrence and severity of DPN in population studies [1]. Spontaneously hypertensive diabetic rats developed a more severe behavioral, physiological and structural phenotype pertinent to clinical DPN [193]. Tissues of neuropathic diabetic patients manifest augmented vasoconstrictive response and diminuted endoneurial blood flow [194]. In turn, vascular deficiency and impaired peripheral nerve perfusion contribute to neural hypoxia and ischemia, two of the well-recognized factors in the pathogenesis of microvascular complica‐ tions in diabetes. This provides a rationale for enhancing vasodilation as a treatment regimen in counteracting diabetes-induced neurovascular stress. This assumption is backed by the observations in experimental diabetes that motor and sensory conduction deficits were normalized by several vasodilating agents with distinct pharmacological actions [195-197]. The most well-established class of compounds in this scenario is angiotensin-converting enzyme (ACE) inhibitors. ACE inhibitors stimulate endothelium-dependent release of nitric oxide and vessel relaxation by antagonizing ACE-mediated formation of the potent vasoconstrictor angiotensin II and deactivation of bradykinin, a strong vasodilator [198]. Combination of these hypotensive effects by ACE inhibitors corrected reductions in nerve blood flow, capillary densities and conduction measurements in STZ-induced diabetic or Zucker fatty rats [199-201].

Although ACE inhibitors are the first line treatment for nephropathy and cardiovascular condition in diabetes [202], there is scarce evidence suggesting the same for diabetic neuro‐ pathy. To date, only one small double-blinded, randomized, placebo-controlled DPN clinical study has been conducted on one ACE inhibitor, trandalapril [203]. In this study, normotensive DPN patients treated with trandalapril over 1 year demonstrated significant improvements in electrophysiological function but not QST, neuropathy symptom/deficit score or autonomic function. A major disappointment came from the Appropriate Blood Pressure Control in Diabetes (ABCD) trial. This prospective study followed 470 type 2 diabetic patients for 5.3 years and found neither moderate nor intensive blood pressure control using nisoldipine (Ca2+ blocker) or enalapril (ACE inhibitor) was effective in modulating the progression of diabetic triopathy (neuropathy, nephropathy, retinopathy) [204]. Furthermore, there were no overall differential outcomes between interventions. This result along with the fact that clinical

respectively.

**2.4. Modulating neurovascular function**

*2.4.1. Nerve blood flow and angiotensin-converting enzyme inhibitors*

In humans, there is no prevailing trend of change in the serum level of NGF in type 2 cohorts with symptomatic DPN [171, 172]. Another study revealed significantly weaker immunoreac‐ tivity of NGF in the lateral calf skin of a group of type 1 diabetics who presented with asymptomatic, early length-dependent loss of nociception and axon reflex vasodilation [173]. However, analysis of the same site from a mixed population of type 1 and type 2 patients with mild early neuropathy indicated that expression of NGF transcripts was higher compared to healthy individuals [174]. Furthermore, epidermal NT-3 protein level markedly increased as a function of the severity of diabetic polyneuropathy [175], whereas CNTF did not vary in postmortem sciatic nerve autopsies between normal and DPN subjects [176]. Likewise, sural nerve IGF-1 mRNA expression was not altered by different durations of DPN [177]. Differing from the findings in animal nerves [161, 178], diabetic humans who developed neuropathy express more trkA and trkC, specific receptors for NGF and NT-3, in the epidermis than those without neuropathy [179]. Whether this reflects a tissue-specific response to diabetes awaits further examination of human nerve biopsies. Clinical testing of recombinant human NGF (rhNGF) perhaps witnessed one of the most spectacular failures in DPN trials. A phase II trial on 250 patients for 6 months reported a robust amelioration on subjective and objective sensory measurements, particularly the components related to small fiber sensory function [180]. When proceeded to a large-scale, multicenter,1-year phase III trial, 1019 participants randomized to receive either placebo or subcutaneous injection of rhNGF could not confirm a neuroprotective effect [181]. Most importantly, severe painful side effects including injection site hyperalgesia and diffuse myalgia significantly limited the tolerable dose to less than 1µg/kg, a dosage 1000 times lower than most of those used in experimental models. This contradicts preclinical data from rodents in which application of NGF reduced pain thresholds [156, 182]. On the opposite side, the observation that NGF evokes pain or hypersensitivity in both animals and humans led to the conception that anti-NGF therapy may reduce neuropathic pain [183-185]. This appeared to be the case in a variety of chronic inflammatory and cancer pain models in which hyperalgesia and/or allodynia were effectively attenuated by antibodies blocking NGF or TrkA [186-188]. In this regard, some proof-of-concept, positive results have been generated in a recent phase III trial on osteoarthritis for a monoclonal antibody against NGF (tanezumab) [189]. However, Pfizer had to temporarily suspend the studies involving DPN after disease worsening and joint replacements occurred in the treatment group.

Other than NGF, a double-blind, placebo-controlled study was also conducted for rhBDNF but found no evidence of improvements on the primary endpoints associated with diabetic sensory neuropathy [190]. Although rhGDNF and rhNT-3 were supposed to enter early clinical assessments for DPN management, they have not yielded any clinical report except the withdrawal of NT-3 from phase I study [156]. It is therefore apparent that the expected outcomes were not met. For IGF-1 and CNTF, development of replacement therapy is also hindered by their non-specific impacts on the central nervous system [191] and muscles [192], respectively.

#### **2.4. Modulating neurovascular function**

of DPN, growth factor therapy early in disease progression may minimize the damage and aid the axonal repair. To this end, abundant support has been produced using an array of spontaneous, chemical or transgenic diabetic models administered recombinant growth factors. Of note, NGF treatment restored neuropeptide level, C-fiber function and dermal myelinated innervation, alleviated neuropathic pain and promoted injury repair [156]. Whereas BDNF and NT-3 elicited a preferential attenuation in the structural and functional changes of large myelinated sensory and motor fibers [14], GDNF and IGF-1 showed a broad preservation of somatic and autonomic nervous system [19, 156]. Moreover, CNTF adminis‐ tration prevented/rescued behavioral and electrophysiological dysfunction, and enhanced

38 Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder

In humans, there is no prevailing trend of change in the serum level of NGF in type 2 cohorts with symptomatic DPN [171, 172]. Another study revealed significantly weaker immunoreac‐ tivity of NGF in the lateral calf skin of a group of type 1 diabetics who presented with asymptomatic, early length-dependent loss of nociception and axon reflex vasodilation [173]. However, analysis of the same site from a mixed population of type 1 and type 2 patients with mild early neuropathy indicated that expression of NGF transcripts was higher compared to healthy individuals [174]. Furthermore, epidermal NT-3 protein level markedly increased as a function of the severity of diabetic polyneuropathy [175], whereas CNTF did not vary in postmortem sciatic nerve autopsies between normal and DPN subjects [176]. Likewise, sural nerve IGF-1 mRNA expression was not altered by different durations of DPN [177]. Differing from the findings in animal nerves [161, 178], diabetic humans who developed neuropathy express more trkA and trkC, specific receptors for NGF and NT-3, in the epidermis than those without neuropathy [179]. Whether this reflects a tissue-specific response to diabetes awaits further examination of human nerve biopsies. Clinical testing of recombinant human NGF (rhNGF) perhaps witnessed one of the most spectacular failures in DPN trials. A phase II trial on 250 patients for 6 months reported a robust amelioration on subjective and objective sensory measurements, particularly the components related to small fiber sensory function [180]. When proceeded to a large-scale, multicenter,1-year phase III trial, 1019 participants randomized to receive either placebo or subcutaneous injection of rhNGF could not confirm a neuroprotective effect [181]. Most importantly, severe painful side effects including injection site hyperalgesia and diffuse myalgia significantly limited the tolerable dose to less than 1µg/kg, a dosage 1000 times lower than most of those used in experimental models. This contradicts preclinical data from rodents in which application of NGF reduced pain thresholds [156, 182]. On the opposite side, the observation that NGF evokes pain or hypersensitivity in both animals and humans led to the conception that anti-NGF therapy may reduce neuropathic pain [183-185]. This appeared to be the case in a variety of chronic inflammatory and cancer pain models in which hyperalgesia and/or allodynia were effectively attenuated by antibodies blocking NGF or TrkA [186-188]. In this regard, some proof-of-concept, positive results have been generated in a recent phase III trial on osteoarthritis for a monoclonal antibody against NGF (tanezumab) [189]. However, Pfizer had to temporarily suspend the studies involving DPN after disease

sensory nerve resprouting in rats previously injected STZ [168, 170].

worsening and joint replacements occurred in the treatment group.

#### *2.4.1. Nerve blood flow and angiotensin-converting enzyme inhibitors*

Multiple epidemiological analyses have previously identified that hypertension strongly increases the occurrence and severity of DPN in population studies [1]. Spontaneously hypertensive diabetic rats developed a more severe behavioral, physiological and structural phenotype pertinent to clinical DPN [193]. Tissues of neuropathic diabetic patients manifest augmented vasoconstrictive response and diminuted endoneurial blood flow [194]. In turn, vascular deficiency and impaired peripheral nerve perfusion contribute to neural hypoxia and ischemia, two of the well-recognized factors in the pathogenesis of microvascular complica‐ tions in diabetes. This provides a rationale for enhancing vasodilation as a treatment regimen in counteracting diabetes-induced neurovascular stress. This assumption is backed by the observations in experimental diabetes that motor and sensory conduction deficits were normalized by several vasodilating agents with distinct pharmacological actions [195-197]. The most well-established class of compounds in this scenario is angiotensin-converting enzyme (ACE) inhibitors. ACE inhibitors stimulate endothelium-dependent release of nitric oxide and vessel relaxation by antagonizing ACE-mediated formation of the potent vasoconstrictor angiotensin II and deactivation of bradykinin, a strong vasodilator [198]. Combination of these hypotensive effects by ACE inhibitors corrected reductions in nerve blood flow, capillary densities and conduction measurements in STZ-induced diabetic or Zucker fatty rats [199-201].

Although ACE inhibitors are the first line treatment for nephropathy and cardiovascular condition in diabetes [202], there is scarce evidence suggesting the same for diabetic neuro‐ pathy. To date, only one small double-blinded, randomized, placebo-controlled DPN clinical study has been conducted on one ACE inhibitor, trandalapril [203]. In this study, normotensive DPN patients treated with trandalapril over 1 year demonstrated significant improvements in electrophysiological function but not QST, neuropathy symptom/deficit score or autonomic function. A major disappointment came from the Appropriate Blood Pressure Control in Diabetes (ABCD) trial. This prospective study followed 470 type 2 diabetic patients for 5.3 years and found neither moderate nor intensive blood pressure control using nisoldipine (Ca2+ blocker) or enalapril (ACE inhibitor) was effective in modulating the progression of diabetic triopathy (neuropathy, nephropathy, retinopathy) [204]. Furthermore, there were no overall differential outcomes between interventions. This result along with the fact that clinical DPN develops and exacerbates in many patients that regularly take the ACE inhibitor casts reasonable doubt on the extent to which ACE intervention is useful in DPN management [205].

*2.4.3. Lipid metabolism and γ-linolenic acid*

γ-linolenic acid as an anti-DPN medicine.

*2.5.1. Reactive oxygen species and α-lipoic acid*

**2.5. Counteracting oxidative stress**

uncontrolled superoxide (O2

−

γ-Linolenic acid is an important precursor for arachidonic acid. The latter produces the potent vasodilator and platelet inhibitor prostacyclin or prostaglandin I2 (PGI2) [219], lack of which can increase the risk of developing thrombosis in diabetic vessels [24] and microvascular diseases. γ-Linolenic acid is primarily synthesized from the dietary ω-6 essential fatty acid linolenic acid, but this reaction is impaired in STZ or alloxan-treated rats [220, 221]. In human type 1 diabetic patients, disturbed fatty acid metabolism has also been inferred from the serum lipid profile [222, 223]. Since γ-linolenic acid also forms the neuronal phospholipids [224, 225], direct supplementation of this polyunsaturated fatty acid can theoretically treat DPN by enhancing both microcirculation and membranous structures in the nervous system, such as the myelin. In keeping with this hypothesis, administration of γ-linolenic acid prevents or reverses the development of experimental DPN in rodents [226-229]. Clinical assessments of the evening primrose oil, the herbal source of γ-linolenic acid, took place in the United Kingdom and suggested an efficacious treatment effect on human DPN [230, 231]. However, some negative outcomes have been obtained for γ-linolenic acid in other clinical conditions by independent groups [232, 233] and the British General Medical Counsel filed a report that the efficacy of evening primrose oil in diabetics claimed by one company-funded trial was falsified [234]. Some issues related to marketing fraud and publication suppression by the drug company attempting to develop evening primrose oil for clinical use have also been raised [235, 236]. Due to these controversies, UK's Medicines Control Agency withdrew the drug's product license. As of today, no further evidence has been acquired to confirm the validity of

From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of…

http://dx.doi.org/10.5772/55364

41

After years of investigations through experimental approaches which harvested knowledge on a plethora of biochemical pathways linking hyperglycemic stress to nerve injury, a general consensus has been reached by the DPN research community that all these complex molecular and cellular events converge on and interact with one universal consequence, oxidative stress [45, 237]. Direct and indirect evidence of oxidative stress in tissue sites of diabetic complications is overwhelming in animals with induced diabetes. In general, hyperglycemia induces a composite oxidative insult to neurons, SCs as well as vasa nervorum through: 1) accelerated free radicals production; 2) increased oxidation and nitration of proteins, lipids and nucleic acids; and 3) deprivation of antioxidant defense system [238]. Many excellent reviews have illustrated and discussed the pathophysiological consequences of redox imbalance in the peripheral nervous system (PNS) [45, 239] therefore an elaborated description will not be provided here. Briefly, increased intracellular glucose metabolism through the classical glycolytic tricarboxylic acid cycle leads to mitochondrial nutrient overload and subsequently

generation of superoxide in conjunction with polyol synthesis exhausts the detoxificating agents including superoxide dismutase and GSH. This eventually gives rise to accumulation

) production by its oxidative respiratory machinery. Excessive

#### *2.4.2. Vascular supply and vascular endothelial growth factor therapy*

Another approach to address vascular insufficiency is to promote the angiogenesis via expression of vascular endothelial growth factor (VEGF), a cytokine primarily mitogenic for vascular endothelial cells. Overexpression of VEGF through gene transfer stimulated vascu‐ larization in both animals [206, 207] and humans [208, 209]. Diabetes was shown to compromise the expression of this growth factor in the skin of patients who also had loss of intraepidermal nerve fiber density (IENFD) [210]. In comparison, most evidence derived from diabetic rodents contradicts with this finding and indicates an upregulation of VEGF in diabetic tissues [211] thatcan be normalized by insulin or NGF infusion [212]. If these observations are true, this could mean VEGF is differentially involved in the pathogenetic processes underlying human and rodent DPN. It is paradoxical, however, that preliminary studies using the same models in which pathological VEGF induction by diabetes was seen also generated data favoring VEGF-enhancing gene therapy in treating DPN. For example, subcutaneous inoculation of herpes simplex virus carrying VEGF-transgene in STZ rats prevented multiple characteristics of experimental DPN, particularly those associated with dorsal sensory function [213]. In a separate report, intramuscular delivery of plasmid DNA encoding VEGF-1 or VEGF-2 completely reversed attenuation of nerve blood flow, slowing of NCV, destruction of vasa nervorum, and dysfunction of small and large fibers in STZ rats [214]. The same study was also able to reproduce the results in rabbits with alloxan-induced diabetes. Two randomized controlled trials (RCTs) have been undertaken to translate this experimental approach to clinical usage. The first trial tested intramuscular VEGF-1 or VEGF-2 gene transfer in 50 DPN patients with presenting symptoms of pain and/or numbness, and achieved an improvement on symptom score, regions of sensory loss and visual analog pain scale over 6-month duration [215]. Other primary and secondary endpoints including quantitative sensory and electro‐ physiological testing were not met. In addition, there were significantly more severe adverse events in gene therapy group compared to placebo group. Among the listed events, hemor‐ rhage, diabetic retinopathy and peripheral edema had been previously brought up as concerns but apparently were not properly addressed during preclinical animal evaluation [216]. The second trial was reported in a published meeting presentation by Sangamo BioSciences, which announced the phase I/II results for a series of injectable plasmids encoding VEGF genetargeting zinc-finger DNA-binding transcription factor with proven-efficacy in experimental models [217]. Of these, SB-509 was praised to be well-tolerated with a most positive outcome in sensory nerve conduction velocity (SNCV), IENFD and neuropathic impairment score. However, the treatment arm as a whole did not obtain a convincing benefit versus placebo to make this a successful trial. With an argument by Sangamo that a carefully chosen cohort may be more sensitive to SB-509, a latest phase IIb study was set to recruit 170 patients with moderate or severe DPN. Despite broad outcome measures and rigorous analysis, the trial was concluded as being unequivocally disappointing which led to the eventual cessation of this Sangamo's lead program [218].

### *2.4.3. Lipid metabolism and γ-linolenic acid*

DPN develops and exacerbates in many patients that regularly take the ACE inhibitor casts reasonable doubt on the extent to which ACE intervention is useful in DPN management [205].

40 Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder

Another approach to address vascular insufficiency is to promote the angiogenesis via expression of vascular endothelial growth factor (VEGF), a cytokine primarily mitogenic for vascular endothelial cells. Overexpression of VEGF through gene transfer stimulated vascu‐ larization in both animals [206, 207] and humans [208, 209]. Diabetes was shown to compromise the expression of this growth factor in the skin of patients who also had loss of intraepidermal nerve fiber density (IENFD) [210]. In comparison, most evidence derived from diabetic rodents contradicts with this finding and indicates an upregulation of VEGF in diabetic tissues [211] thatcan be normalized by insulin or NGF infusion [212]. If these observations are true, this could mean VEGF is differentially involved in the pathogenetic processes underlying human and rodent DPN. It is paradoxical, however, that preliminary studies using the same models in which pathological VEGF induction by diabetes was seen also generated data favoring VEGF-enhancing gene therapy in treating DPN. For example, subcutaneous inoculation of herpes simplex virus carrying VEGF-transgene in STZ rats prevented multiple characteristics of experimental DPN, particularly those associated with dorsal sensory function [213]. In a separate report, intramuscular delivery of plasmid DNA encoding VEGF-1 or VEGF-2 completely reversed attenuation of nerve blood flow, slowing of NCV, destruction of vasa nervorum, and dysfunction of small and large fibers in STZ rats [214]. The same study was also able to reproduce the results in rabbits with alloxan-induced diabetes. Two randomized controlled trials (RCTs) have been undertaken to translate this experimental approach to clinical usage. The first trial tested intramuscular VEGF-1 or VEGF-2 gene transfer in 50 DPN patients with presenting symptoms of pain and/or numbness, and achieved an improvement on symptom score, regions of sensory loss and visual analog pain scale over 6-month duration [215]. Other primary and secondary endpoints including quantitative sensory and electro‐ physiological testing were not met. In addition, there were significantly more severe adverse events in gene therapy group compared to placebo group. Among the listed events, hemor‐ rhage, diabetic retinopathy and peripheral edema had been previously brought up as concerns but apparently were not properly addressed during preclinical animal evaluation [216]. The second trial was reported in a published meeting presentation by Sangamo BioSciences, which announced the phase I/II results for a series of injectable plasmids encoding VEGF genetargeting zinc-finger DNA-binding transcription factor with proven-efficacy in experimental models [217]. Of these, SB-509 was praised to be well-tolerated with a most positive outcome in sensory nerve conduction velocity (SNCV), IENFD and neuropathic impairment score. However, the treatment arm as a whole did not obtain a convincing benefit versus placebo to make this a successful trial. With an argument by Sangamo that a carefully chosen cohort may be more sensitive to SB-509, a latest phase IIb study was set to recruit 170 patients with moderate or severe DPN. Despite broad outcome measures and rigorous analysis, the trial was concluded as being unequivocally disappointing which led to the eventual cessation of this

*2.4.2. Vascular supply and vascular endothelial growth factor therapy*

Sangamo's lead program [218].

γ-Linolenic acid is an important precursor for arachidonic acid. The latter produces the potent vasodilator and platelet inhibitor prostacyclin or prostaglandin I2 (PGI2) [219], lack of which can increase the risk of developing thrombosis in diabetic vessels [24] and microvascular diseases. γ-Linolenic acid is primarily synthesized from the dietary ω-6 essential fatty acid linolenic acid, but this reaction is impaired in STZ or alloxan-treated rats [220, 221]. In human type 1 diabetic patients, disturbed fatty acid metabolism has also been inferred from the serum lipid profile [222, 223]. Since γ-linolenic acid also forms the neuronal phospholipids [224, 225], direct supplementation of this polyunsaturated fatty acid can theoretically treat DPN by enhancing both microcirculation and membranous structures in the nervous system, such as the myelin. In keeping with this hypothesis, administration of γ-linolenic acid prevents or reverses the development of experimental DPN in rodents [226-229]. Clinical assessments of the evening primrose oil, the herbal source of γ-linolenic acid, took place in the United Kingdom and suggested an efficacious treatment effect on human DPN [230, 231]. However, some negative outcomes have been obtained for γ-linolenic acid in other clinical conditions by independent groups [232, 233] and the British General Medical Counsel filed a report that the efficacy of evening primrose oil in diabetics claimed by one company-funded trial was falsified [234]. Some issues related to marketing fraud and publication suppression by the drug company attempting to develop evening primrose oil for clinical use have also been raised [235, 236]. Due to these controversies, UK's Medicines Control Agency withdrew the drug's product license. As of today, no further evidence has been acquired to confirm the validity of γ-linolenic acid as an anti-DPN medicine.

#### **2.5. Counteracting oxidative stress**

#### *2.5.1. Reactive oxygen species and α-lipoic acid*

After years of investigations through experimental approaches which harvested knowledge on a plethora of biochemical pathways linking hyperglycemic stress to nerve injury, a general consensus has been reached by the DPN research community that all these complex molecular and cellular events converge on and interact with one universal consequence, oxidative stress [45, 237]. Direct and indirect evidence of oxidative stress in tissue sites of diabetic complications is overwhelming in animals with induced diabetes. In general, hyperglycemia induces a composite oxidative insult to neurons, SCs as well as vasa nervorum through: 1) accelerated free radicals production; 2) increased oxidation and nitration of proteins, lipids and nucleic acids; and 3) deprivation of antioxidant defense system [238]. Many excellent reviews have illustrated and discussed the pathophysiological consequences of redox imbalance in the peripheral nervous system (PNS) [45, 239] therefore an elaborated description will not be provided here. Briefly, increased intracellular glucose metabolism through the classical glycolytic tricarboxylic acid cycle leads to mitochondrial nutrient overload and subsequently uncontrolled superoxide (O2 − ) production by its oxidative respiratory machinery. Excessive generation of superoxide in conjunction with polyol synthesis exhausts the detoxificating agents including superoxide dismutase and GSH. This eventually gives rise to accumulation of other reactive oxygen (ROS) and nitrogen species (RNS) such as hydrogen peroxide (H2O2), hydroxyl radicals (OH•) and peroxynitrite (NO•). Other hyperglycemia-initiated events such as AGE formation and NGF deficiency have also been suggested to fuel the ROS generation in various compartments. These highly reactive free radicals can non-specifically oxidize and nitrosylate cellular/extracellular biomolecules and undermine organellar function. Particular‐ ly, increased protein nitration, lipid peroxidation products and mitochondria dysfunction are predominant phenomena in DRGs and sciatic nerves in diabetic animals [240-242]. Compared to the clear evidence of oxidative damage in experimental DPN, expression of the correspond‐ ent biomarkers indicating oxidative stress in human tissues is rather vague [239, 243]. Some studies even suggested a reduced free radical reaction in diabetic patients versus normal control [244, 245]. Further, despite a strong rationale and the promise of substantial neuro‐ protection by anti-oxidant treatments in rodent diabetics [246-249], this anti-oxidative ap‐ proach is not spared from the irreproducibility of the results obtained from basic research in clinical practice.

damage has been equivocal and usually halted at human trials due to toxicity, lack of efficacy or both (Figure 1). Clearly, the pharmacological translation from our decades of experimental modeling to clinical practice with regard to DPN has thus far not even close to satisfactory. Undoubtedly, the flawed design of some clinical trials has led to the inadequate evaluation of certain candidate compounds and for a thorough discussion on this specific topic the readers are referred elsewhere [256]. In this section, we focus on discussing some of the fundamental

From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of…

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43

Whereas a majority of the drugs investigated during preclinical testing executed experimen‐ tally desired endpoints without revealing significant toxicity, more than half that entered clinical evaluation for treating DPN were withdrawn as a consequence of moderate to severe adverse events even at a much lower dose. Generally, using other species as surrogates for human population inherently encumbers the accurate prediction of toxic reactions for several

First of all, it is easy to dismiss drug-induced non-specific effects in animals—especially for laboratory rodents who do not share the same size, anatomy and physical activity with humans. Events such as cardiac attack are often overlooked without a complex and careful examination. A case in point is the anti-diabetic drug Avandia for which the market approval has been a center of dispute. Avandia's active ingredient rosiglitazone promotes insulin sensitivity by activating peroxisome proliferator-activated receptors (PPARs) and was claimed by its maker GlaxoSmithKline to be safe in the preclinical report. Some even went further to advocate the favorable application of rosiglitazone to heart conditions based on its positive influence on cardiovascular biomarkers in rodent studies [257, 258]. Only after accumulating incidents of congestive heart failure among patients receiving Avandia was presented to the FDA, did it begin to spur wide concerns and active investigations of the serious cardiotoxicity

Second, some physiological and behavioral phenotypes observable in humans are impossible for animals to express. In this aspect, photosensitive skin rash and pain serve as two good examples of non-translatable side effects. Rodent skin differs from that of humans in that it has a thinner and hairier epidermis and distinct DNA repair abilities [260]. Therefore, most rodent stains used in diabetes modeling provide poor estimates for the probability of cutane‐ ous hypersensitivity reactions to pharmacological treatments [261]. Although skin engraft‐ ment onto nude mice has been attempted to circumvent this issue [260], mice with immunodeficiency do not constitute an appropriate background for studying diabetes. Another predicament is to assess pain in rodents. The reason for this is simple: these animals cannot tell us when, where or even whether they are experiencing pain, leaving us to read. Since there is not any specific type of behavior to which painful reaction can be unequivocally associated, this often leads to underestimation of painful side effects during preclinical drug

The third problem is that animals and humans have different pharmacokinetic and toxicologi‐ cal responses. For instance, troglitazone (Rezulin), another anti-hyperglycemic PPAR agonist,

species differences that render a direct translation unrealistic.

**3.1. Failure to predict toxic effects**

by Avandia in humans and animals [259].

screening (e.g. rhNGF).

reasons.

Among a number of anti-oxidants that corrected experimental DPN, α-lipoic acid (ALA) has gone the furthest into clinical use, while the others have proven largely ineffective [14, 250]. ALA or thioctic acid is naturally synthesized in mitochondria and has a powerful antioxidant capacity because of its dual ability to scavenge ROS/transition metals and regenerate other endogenous antioxidants. Approximately 7 double-masked multicenter RCTs, including the series of ALADIN, SYDNEY and NATHAN, testing the efficacy of ALA in treating sympto‐ matic DPN have been completed in Europe [251]. Of these, a general benefit on sensory symptoms and deficits was extrapolated by a meta-analysis incorporating 4 trials (ALADIN I, ALADIN III, SYDNEY, NATHAN II) that treated subjects with 600 mg/day ALA via intravenous infusion for 3 weeks [252]. However, there is an overall mixed bag of results and several therapeutically important indices including symptoms score, nerve conduction and QST were not consistently ameliorated in these studies [205, 252, 253]. Notably, some asserted improvement fell below the clinically meaningful threshold of 30% when adjusted to placebo control [254]. It is also discouraging that trials in which patients received oral dosing of ALA presented only marginal benefit; this significantly precludes the oral application of ALA. Although ALA has been marketed in Germany for treating DPN and is available as nutritional supplement in the US, current existing evidence suggests that ALA at best only retards the neuropathic progression in diabetes.
