Open access peer-reviewed chapter

# From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of Diabetic Peripheral Neuropathy

By Chengyuan Li, Anne E. Bunner and John J. Pippin

Submitted: July 9th 2012Reviewed: December 3rd 2012Published: March 27th 2013

DOI: 10.5772/55364

## 1. Introduction

### 3.1. Failure to predict toxic effects

Whereas a majority of the drugs investigated during preclinical testing executed experimentally 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endpoints without revealing significant toxicity, more than half that entered clinical evaluation for treating DPN were withdrawn as a consequence of moderate to severe adverse events even at a much lower dose.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 reasons.

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 by Avandia in humans and animals [259].

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 cutaneous hypersensitivity reactions to pharmacological treatments [261]. Although skin engraftment 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 screening (e.g. rhNGF).

The third problem is that animals and humans have different pharmacokinetic and toxicological responses. For instance, troglitazone (Rezulin), another anti-hyperglycemic PPAR agonist, was withdrawn after inducing idiosyncratic liver failure in patients but a similar hepatotoxicity could not be reproduced in animal models [262, 263]. Even in organ systems that were previously defined as having an overall high rate of interspecies toxicity concordance, unanticipated drug toxicity can still occur. This was the case for trastuzumab (Herceptin), a humanized monoclonal antibody that treats advanced breast carcinoma by binding and blocking human epidermal growth factor receptor 2 (HER2). Both preclinical and on-going toxicological studies in rhesus monkeys and rodents indicated no evidence of cardiac dysfunction [264]. However, trastuzumab administration to patients during clinical trials caused frequent and severe cardiomyopathy [265]. As discussed in a published scientific document of Herceptin toxicity by the European Medicines Agency, it is also unsuitable to assess the cytotoxicity of this antibody that specifically recognizes a single human protein in nonhuman species which have a distinct molecular and immunogenic environment [264]. In addition to the inaccuracies, disparities in pharmacokinetics underpin some of the extreme species differences. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neurotoxicity is a classic example. MPTP becomes poisonous to dopaminergic neurons once metabolized to MPP+ by the enzyme monoamine oxidase-B (MAO-B) and elicits permanent Parkinson-like symptoms in human subjects [266]. In sharp contrast, MPTP is barely psychoactive in rats since they produce minimal MPP+ and only mild damage to mouse brains due to much faster clearance of MPP+ compared to primates [267]. By the same token, 350 mg of aspirin can be eliminated by half from human circulation in about 3 hours but retained in feline plasma for 37.5 hours, which is essentially lethal to these animals [268]. The argument can be finally strengthened by the work of two independent groups, who compared bioavailability between primates, rodents and dogs for various drugs and both demonstrated that no correlation exists between animal and human data [269]. The matter of drug-induced non-specific effects and uniquely human phenotypes may theoretically be resolved via rigorous pathological evaluation and better experimental method. By comparison, the pharmacokinetic and toxicological data highlights profound interspecies barriers and may not succumb to current technical manipulation. Considering some of the drugs were withdrawn when unexpected toxicological outcomes occur in only 1-2% of the population, relying on laboratory models to predict drug safety certainly puts us in a dilemma with very little medical and ethical risks from which our society can suffer (Figure 1).

### 3.2. Failure to recapitulate human neuropathologies

Genetic or chemical-induced diabetic rats or mice have been a major tool for preclinical pharmacological evaluation of potential DPN treatments. Yet, they do not faithfully reproduce many neuropathological manifestations in human diabetics. The difficulty of such begins with the fact that it is not possible to obtain in rodents a qualitative and quantitative expression of the clinical symptoms that are frequently presented in neuropathic diabetic patients, including spontaneous pain of different characteristics (e.g. prickling, tingling, burning, squeezing), paresthesia and numbness. As symptomatic changes constitute an important parameter of therapeutic outcome, this may well underlie the failure of some aforementioned drugs in clinical trials despite their good performance in experimental tests measuring behavioral responses of animals to external stimuli (Table 1). Development of nerve dysfunction in diabetic rodents also does not follow the common natural history of human DPN. As described earlier, sensory neuropathy in humans typically adopts a length-dependent, “stocking-glove” loss of sensation that slowly progresses from distal to proximal. Such a pattern was never functionally recapitulated in the commonly used type 1 and type 2 diabetic animal models, including STZ-injected rats, Zucker diabetic fatty (ZDF) rats and db/db mice. Besides the lack of anatomical resemblance, the changes in disease severity are often missing in these models. For example, although the majority of diabetic rodent models developed thermal hypoalgesia with long durations of diabetes as revealed by the sensory assay correspondent to that of QSTs in humans, there is no agreement between different studies in a consistent trend of progressive decline in thermal pain perception [270-272], a well-known phenomenon in patients. Alterations in thermal sensation in the tails of diabetic rodents varied upon studies and species used [273-275] and several groups have documented increased temperature perception after prolonged diabetes [276, 277], thus falsifying the relevance of tail flick test to human conditions. More importantly, foot ulcers that occur as a late complication to 15% of all individuals with diabetes [14] do not spontaneously develop in hyperglycemic rodents. Superimposed injury by experimental procedure in the foot pads of diabetic rats or mice may lend certain insight in the impaired wound healing in diabetes [278] but is not reflective of the chronic, accumulating pathological changes in diabetic feet of human counterparts. Another salient feature of human DPN that has not been described in animals is the predominant sensory and autonomic nerve damage versus minimal involvement of motor fibers [279]. This should elicit particular caution as the selective susceptibility is critical to our true understanding of the etiopathogenesis underlying distal sensorimotor polyneuropathy in diabetes. In addition to the lack of specificity, most animal models studied only cover a narrow spectrum of clinical DPN and have not successfully duplicated syndromes including proximal motor neuropathy and focal lesions [279].

Morphologically, fiber atrophy and axonal loss exist in STZ-rats and other diabetic rodents but are much milder compared to the marked degeneration and loss of myelinated and unmyelinated nerves readily observed in human specimens [280]. Of significant note, rodents are notoriously resistant to developing some of the histological hallmarks seen in diabetic patients, such as segmental and paranodal demyelination [44]. There are sporadic reports of demyelination in STZ and genetically diabetic Bio-Breeding (BB) rats after 8-12 months of diabetes [58, 281-283]. However, this is apparently related to a different microvascular pathology as morphometric analysis of sural and tibial vasa nervorum in these rats revealed dilated lumina, flattening of endothelial cells and microvessel walls [284], contrasting with the basement membrane thickening, endothelial hyperplagia and narrowing of endoneurial lumen in human diabetics [285, 286]. Similarly, the simultaneous presence of degenerating and regenerating fibers that is characteristic of early DPN has not been clearly demonstrated in these animals [44]. Since such dynamic nerve degeneration/regeneration signifies an active state of nerve repair and is most likely to be amenable to therapeutic intervention, absence of this property makes rodent models a poor tool in both deciphering disease pathogenesis and designing treatment approaches. Given that our ability to devise a cure for human DPN depends ultimately on our successful understanding and reduction of its various functional and structural indexes, failure of most animal models to replicate these human neuropathologies with high fidelity renders this task difficult at best.

### Table 1.

Comparison of DPN Characteristics between Humans and Frequently Used Laboratory Rodent Models

Abbreviations: NOD=non-obese diabetic, AR=aldose reductase

### 3.3. Overrepresentation of pathogenetic pathways

STZ is a glucose analog of selective toxicity to pancreatic β-cells and induces insulin-deficiency and hyperglycemia mimicking that in human type 1 diabetes mellitus. Injection of this chemical provides a convenient and affordable tool in inducing robust hyperglycemia in animals with good control over disease onset and duration. Therefore, STZ-rats have been favored by researchers during preclinical drug assessments for diabetic complications [280]. However, STZ typically produces a rather immediate, severe hypoinsulinemia and elevation of blood glucose, whereas the development of hyperglycemia in most human conditions is slow and modest [287]. The contrariety manifests stably in the serum HbA1c levels. While the non-diabetic range (~4-5.6%) is similar, a single administration of STZ to Wistar rats can increase the HbA1c to above 12% in 4-5 weeks [288, 289], which indicates a very poor glucose control that is considered rare in the clinic setting with anti-diabetic care. In fact, less than 15% of patients may have an HbA1c level exceeding 9% by sample estimation [290]. Such extreme hyperglycemia in STZ-treated rats could give rise to exaggerated glucose accumulation and metabolic derangements that would not be commonly present in human diabetics. Indeed, the concentrations of sorbitol and fructose per unit weight of nerve tissue in STZ diabetic rats is consistently increased and dramatically higher in comparison with human diabetics, who on average also do not uniformly show upregulation of these glucose metabolites via polyol pathway [44, 55, 79]. Of interesting note, under normal physiological conditions the contents of nerve sorbitol in rodents are almost 10-fold higher than those in humans, suggesting some species difference in the relative involvement of AR in glucose metabolism during both normo- and hyperglycemia. Observations of polyol pathway utilization in different species and cell types vary widely; the total glucose utilization through polyol pathway is one third in rabbit ocular lenses and only one tenth in human erythrocytes in response to high glucose stress [45, 291]. Consistent with an inverse association between increased polyol flux and electrophysiological dysfunction, diabetic rodents frequently exhibit 10 m/s or more reduction in NCV within the typical 6-20 week experimental duration [271, 292-294]. By contrast, the deterioration of NCV in human patients gradually takes place and has an average loss of 0.5 m/s per year [1] (Table 1). It is also suspicious that the profound and precipitated NCV deceleration in STZ-rodents occur without apparent histopathological changes, which can be a prominent feature in diabetic neuropathic patients at early stage. Therefore, enhanced AR activity might contribute differently or less significantly to the pathogenesis of DPN in humans than rodents. This could explain why AR inhibitors, and by extension, many other pathogenetically targeted inhibitors afford potent neuroprotection in experimental studies but only marginal effects in clinical trials.

Another criticism is that most STZ models were rendered diabetic at puberty since administering STZ to rodents after sexual maturation cannot always produce peripheral nerve abnormalities [280, 295]. Unlike matured nerves that displayed little change in response to diabetic insults, immature peripheral nerves readily manifest hyperglycemia-induced morphological and electrophysiological deficits within an even shorter duration [295]. However, such a phenotype bears little relevance to 90% of clinical conditions, in which diabetes-induced nerve damage has an adult onset and slow time course.

### 3.4. Other physical and environmental factors

Humans certainly share considerable biological similarities with other mammals. In the nervous system, these include some of the nociceptive responses and higher cognitive activities. At the same time, no one would suggest that humans and animals are the same—they obviously differ in many physiological and behavioral aspects. The question is: can we obtain effective therapeutic applicability after evolution has well separated our species from others? In order to answer this, it is necessary to carefully examine these differences and their impacts on the pharmacokinetic and pharmacological extrapolation. As delineating every single molecular, cellular and phenotypic difference is a laborious task, we will highlight only those relevant to our discussion of DPN. When comparing humans with the conventionally used experimental animals, namely rats and mice, the most conspicuous difference is anatomical. With particular respect to neuroanatomy, a peripheral axon in humans can reach as long as one meter [296] whereas the maximal length of the axons innervating the hind limb is five centimeters in mice and twelve centimeters in rats. This short length makes it impossible to study in rodents the prominent length dependency and dying-back feature of peripheral nerve dysfunction that characterizes human DPN. Even if size were an issue and macro-structure appears similar, there might still be striking differences in the micro-structure within the tissue or organ. This is the case for insulin-secreting islets. For decades the cytoarchitecture of human islets was assumed to be just like those in rodents with a clear anatomical subdivision of β-cells and other cell types. By using confocal microscopy and multi-fluorescent labeling, it was finally uncovered that human islets have not only a substantially lower percentage of β-cell population, but also a mixed—rather than compartmentalized—organization of the different cell types [297]. This cellular arrangement was demonstrated to directly alter the functional performance of human islets as opposed to rodent islets. Although it is not known whether such profound disparities in cell composition and association also exist in the PNS, it might as well be anticipated considering the many sophisticated sensory and motor activities that are unique to humans.

Considerable species difference also manifest at a molecular level. The chemical structure and signaling profile of a molecule may not always be conserved throughout the evolution. Such difference, although small, can account for a significant translational limitation for pharmacological treatments targeted at a specific biomolecule. A good explanation is the case of trastuzumab. As mentioned earlier, trastuzumab was specifically designed to immuno-antagonize HER2, thereby inhibiting cancer cell growth. However, this drug could not be adequately assessed in rodents or primates because of the inability of this human protein-targeting antibody to recognize the HER2 homologues expressed in these nonhuman species [264]. Despite the successful employment of nude mice for the preclinical evaluation of trastuzumab, a comprehensive pharmacological and pharmacokinetic profile was not obtained for this humanized antibody and it resulted in unpredicted toxicity in patients. While the molecular difference might not be as serious of a problem for rhNGF and rhVEGF, critical retrospective examination into this aspect may lend some insight into the failure of these gene therapies in DPN trials. At least 80% of human genes have a counterpart in the mouse and rat genome. However, temporal and spatial expression of these genes can vary remarkably between humans and rodents, in terms of both extent and isoform specificity. The first is evident from the differential level of MAO-B expression in humans and rats which resulted in distinct susceptibility of these two species to MPTP-induced neurotoxicity [266]. The second category involves protein families comprising multiple isoforms owing to different promoter usage and alternative gene splicing. For instance, the enzyme PKC has at least 12 different subtypes, of which, PKC-α is predominantly expressed in human hearts and PKC-ε in rodents [298]. Since activation of PKC-α and PKC-ε are differentially regulated, species-specific PKC inhibitors will need to be developed in order to efficiently block the pathogenic action of this kinase in cardiomyopathy, especially when a non-selective inhibition of PKC function is unwanted or even detrimental. Given that the efficacy of ruboxistaurin in treating DPN was also based on data from rat diabetic models [150, 151], it is imperative to speculate that the unsatisfactory results of ruboxistaurin in patients is due at least in part to a relatively less important role of PKC-β in the pathological development of diabetic human nerves. The last type of molecular difference is that the components along a particular signaling axis may be preferentially vulnerable to pathological alteration in different species. This possibility has been largely ignored but could underpin a major limitation in current translational research. One typical example is that much has been learned regarding the anti-hyperphagic effects of leptin from ob/ob mice, which also led to the exciting finding that administration of this hormone can successfully suppress weight gain [299]. Nonetheless, this offered little treatment benefit for the majority of obese people (99.95%) who have impaired signaling downstream of leptin instead of leptin deficiency as observed in ob/ob mice [300]. Some may argue that these issues can be overcome by creating genetically engineered or “humanized” mice in which a mouse gene is substituted by the human version. However, transgenic or knockout mice can be afflicted with developmental deficits and alterations which are inappropriate for modeling a chronic disease that appears in the later life time, such as type 2 diabetes and its complications. Moreover, we do not know whether a genetically introduced human protein—if it is different enough from the murine orthologue that a transgene is necessary—faithfully maintains the same expression and interaction properties in mouse system as it would in humans.

Ultimately, a fundamental problem associated with resorting to rodents in DPN research is to study a human disorder that takes decades to develop and progress in organisms with a maximum lifespan of 2-3 years. The longest duration of experimental diabetes in a rodent model was documented by Ras et al., who observed leptin-deficient db/db mice for 17 months and reported only mild pathological changes in the peripheral nerve fibers [301]. It is thus fair to say that a full clinical spectrum of the maturity-onset DPN likely requires a length of time exceeding the longevity of rodents to present and diabetic rodent models at best only help illustrate the very early aspects of the entire disease syndrome. Since none of the early pathogenetic pathways revealed in diabetic rodents will contribute to DPN in a quantitatively and temporally uniform fashion throughout the prolonged natural history of this disease, it is not surprising that a handful of inhibitors developed against these processes have not benefited patients with relatively long-standing neuropathy. As a matter of fact, any agents targeting single biochemical insults would be too little too late to treat a chronic neurological disorder with established nerve damage and pathogenetic heterogeneity (Figure 2). In DPN, such heterogeneity is the consequence of a complex interplay between genetic predisposition, physical characteristics, nutritional and other environmental factors. On the contrary, experimental rodents are maintained at a homogeneous genetic background. Genetic homogeneity becomes particularly apparent with the inbred strains and genetically engineered mice, making them more of a tool to elucidate the contribution of a specific component to disease development and less of a tool for an accurate prediction of the likelihood that a treatment will be effective for a general population. Apart from these internal factors, laboratory caged animals have an uniform dietary constitution, life cycle and environmental contact, therefore would not be exposed to the majority of the external risk factors otherwise incurred by individual patients, such as smoking and alcohol consumption [10]. Finally, humans have some unique behaviors that assume an integral part of DPN-associated complications but cannot be adopted by animals. This is perhaps the simplest reason why diabetic rodents are immune to gangrenous foot ulceration as upright walking has not evolved in these species.

## 4. Conclusion and outlook

Needless to say, DPN has been a significant source of diabetes-induced mortality and morbidity that strike individuals, families and society with a staggering health and economic cost. There is little doubt that the need for effective DPN management is currently unmet and better therapeutic regimens ought to be sought. The invasive nature of present methods of biochemical, structural and functional measurements dictates that systemic and longitudinal assessments are not feasible in humans. To address this, miscellaneous rodent models have been created and used as substitutes for diabetic patients for the purpose of uncovering the pathogenetic mechanisms and testing potential pharmacological treatments. However, these conventional approaches have so far failed to yield a successful therapeutic translation. Further, animal surrogates are afflicted with species differences in genotype and behavior, nerve structure and metabolism, duration of diabetes, and tissue vulnerability, which allow limited transferability of animal results into clinical settings. It is important to point out that the present review does not argue against the ability of animal models to shed light on basic molecular, cellular and physiological processes that are shared among species. Undoubtedly, animal models of diabetes have provided abundant insights into the disease biology of DPN. Nevertheless, the lack of any meaningful advance in identifying a promising pharmacological target necessitates a reexamination of the validity of current DPN models as well as to offer a plausible alternative methodology to scientific approaches and disease intervention. After a critical reevaluation of the experimental results and clinical outcomes for several previously high-profile anti-DPN drugs, we conclude that the fundamental species differences have led to misinterpretation of rodent data and overall failure of pharmacological investment. As more is being learned, it is becoming prevailing that DPN is a chronic, heterogeneous disease unlikely to benefit from targeting specific and early pathogenetic components revealed by animal studies. Rather, an efficacious therapy must impact on multiple etiologic events and manage various risk factors. In this regard, rigorous lifestyle modulation may simultaneously intervene with a multitude of internal and external diabetogenic processes without generating significant tissue toxicity and side effects. Particularly, diet and exercise intervention provides an approach to improve metabolic management and enhance long-term reparative and regenerative capacity of diabetic nerves. Moreover, investigating the disease process via human-based study to the extent possible promises to lend much better insight into the pathology and pathogenesis of DPN as well as the clinical utility of potential treatments. We propose that future research should put an emphasis on advancing methodological and technological approaches that maximizes the access and utilization of human specimens under ethical guidelines, and on refining lifestyles for preventing and modifying DPN, which are more cost-effective and directly applicable to clinical practice in this otherwise largely intractable disorder.

## Acknowledgments

This work is supported and funded by the Physicians Committee for Responsible Medicine. The authors thank Dr. Neal Barnard and Dr. Charu Chandrasekera for their help and expertise on this paper.

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© 2013 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Chengyuan Li, Anne E. Bunner and John J. Pippin (March 27th 2013). From Animal Models to Clinical Practicality: Lessons Learned from Current Translational Progress of Diabetic Peripheral Neuropathy, Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder, Nizar Souayah, IntechOpen, DOI: 10.5772/55364. Available from:

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