Topical Atorvastatin in the Treatment of Diabetic Wounds

BASIC INVESTIGATION

Topical Atorvastatin in the Treatment of Diabetic Wounds Serdar Toker, MD, Erim Gulcan, MD, Muhammet Kasım C ¸ aycı, PhD, Esra G. Olgun, MD, ¨ Enver Erbilen, MD and Yusuf Ozay, PhD

Abstract: Background: Currently, it is reported that statins may be useful in the treatment of diabetes mellitus foot ulceration. The aim of this study was to evaluate treatment of the wounds in streptozotocininduced diabetic rats with local atorvastatin. Methods: Two 15 ⫻ 15 mm-sized wounds were created in 28 streptozotocin-induced rats. A total of 56 diabetic wounds were studied in 8 groups (n ⫽ 7). No treatment was administered in the first and second groups, which lasted for 7 and 14 days, respectively. Third and fourth groups consisted of diabetic rats that were administered 1:1 mixture of lanolin and vaseline therapy for 7 and 14 days, respectively. One percent statin plus 1:1 mixture of lanolin and vaseline was used in the fifth and sixth groups for 7 and 14 days, respectively; and in seventh and eighth groups, 5% statin plus 1:1 mixture of lanolin and vaseline therapy was used for 7 and 14 days, respectively. On the 7th and 14th days, state of the wound healing was observed, and the percent of wound healing was determined by measuring its size and by performing a histopathologic study. The statistical analyses were performed by Mann–Whitney U test, using SPSS 14.0 software. Results: On the 14th day, the rates of wound healing in the first, second, third, and fourth groups were 14%, 40%, 96.59%, and 96.51%, respectively. This ratio was calculated by the formula healing ratio (%) ⫽ 100 ⫻ (1⫺wound area/initial wound area). Accordingly, in the multiple comparisons, the rates of wound healing were found to be significantly higher in the diabetic rat groups administered 1% and 5% atorvastatin compared with those administered a mixture of lanolin-vaseline and the untreated group (for comparison each one P ⬍ 0.001). Conclusions: Local atorvastatin therapy may be useful for healing the wounds in diabetic rats. Further clinical and experimental studies are needed to confirm these results. Key Indexing Terms: Statins; Diabetic ulcer; Wound healing; Streptozotocin-induced diabetes. [Am J Med Sci 2009;338(3):201–204.]

W

ound healing is a dynamic and complex process that involves a well-coordinated, highly regulated series of events, including inflammation, tissue formation, revascularization, and tissue remodeling. However, this orderly process is impaired in certain pathologic conditions, such as diabetes mellitus (DM),1 making the diabetic wounds a great problem for health services. Because the diabetic wounds do not heal properly, this problem becomes a source of major suffering and economic constraint. The most common site for diabetic patients to have a diabetic wound is the lower extremities and especially the feet. According to recent reports, only two thirds of diabetic foot ulcers heal and up to 28% may result in amputation.2 The pathogenesis of diabetic foot ulcers is complex, and it is well-known that a number of

From the Departments of Orthopaedics and Traumatology (ST) and Internal Medicine (EG), School of Medicine, Dumlupinar University, Kutahya, Turkey; Department of Biology (MKC), Faculty of Arts and Science, Dumlupinar University, Kutahya, Turkey; Departments of Pathology (EGO) and Cardiology (EE), School of Medicine, Dumlupinar University, Kutahya, Turkey; and Health College (YO), Kırsehir Ahı Evran University, Kirsehir, Turkey. Submitted March 8, 2009; accepted in revised form April 8, 2009. Correspondence: Serdar Toker, MD, Department of Orthopaedics and Traumatology, Dumlupinar University, Tavsanlı yolu, 43270 Kutahya, Turkey (E-mail: [email protected]).

contributory factors working together ultimately lead to impaired healing. Several factors, such as peripheral neuropathy, peripheral vascular disease, and peripheral edema, have been identified as the commonest factors responsible for impaired healing after trauma.3 Because DM is growing at epidemic proportions worldwide, the prevalence of diabetes-related complications is bound to increase.4 The lifetime incidence of a diabetic developing a foot ulcer has been estimated at 15% to 25%.5,6 Patients can undergo surgical interventions, such as peripheral bypass, to improve vascular circulation; wound debridement to control the wound; and amputation, if required. However, medical therapies for wound care are limited, therefore, development of new treatment modalities to improve wound healing in diabetic patients is an essential and emerging field of investigation.7 Numerous conservative methods, such as honey as a dressing solution,8 topical antimicrobial therapies,9 total contact casting,10 wound dressings,11 extracorporeal shock wave therapy,12 vacuum-assistant closure,13 for the treatment of diabetic wounds have been reported in the literature. Herbal therapies were also reported.14 In this study, a well-known agent, statin, is used for the treatment of diabetic wounds. Currently, it is reported that statins may be useful in the treatment of diabetic foot ulceration (DFU).15 However, this agent has never been used in an experimental study focused on diabetic wounds. We believe that this study provides an innovation in diabetic wound treatment.

MATERIALS AND METHODS Animals Albino Wistar rats of either sex weighing 200 to 250 g obtained from the central animal house of Dumlupinar University were used for the study. Rats were housed individually in cages, maintained under standard conditions (12-hour light/dark cycle; 25 ⫾ 3°C), and fed with standard pellet and water ad libitum. Animal studies were performed after approval from the Animal Care and Ethics Committee of Medical Faculty of Dumlupinar University, and the “Principles of laboratory animal care” (NIH publication no. 85–23, received 1985) guidelines were followed. Preparation of Statin Cream Lanolin and vaseline were used to formulate a statin cream base. For 1% and 5% statin, statin powder was dissolved in warm water and then mixed with cream base during the cream-forming process.16 Streptozotocin-Induced Diabetes Diabetes was induced by a single-dose intraperitoneal injection of streptozotocin (65 mg/kg; Sigma-Aldrich, St. Louis, MO) freshly prepared in saline.17 Three days after streptozotocin injection, glycemia was confirmed, and rats showing fasting blood glucose higher than 250 mg/dL were considered as diabetic rats. Subsequently, animals were randomly divided into 4 groups, and each group consisted of 14 rats.

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Determination of Blood Glucose Capillary blood glucose was measured with glucose test strips (IME-DC Blood Glucose Meter System, Int. Medical Equipment Diabetes Care, Germany). Induction of Skin Ulcer A deep skin ulcer model was created using diabetic rats as follows.16 The rats were anesthetized by intraperitoneal injection of xylazin hydrochloride (10 mg/kg) and ketamine hydrochloride (25 mg/ kg), and their back hair was shaved and the application field was outlined with a marking pen just before removing the skin. Two full-thickness skin wounds in each rat were created by removing the skin in a circle with 1.5 cm diameter on the dorsum. A total of 56 diabetic wounds were studied in 8 groups (n ⫽ 7). No treatment was administered in the first and second groups, which lasted for 7 and 14 days, respectively. Third and fourth groups consisted of diabetic rats that were administered 1:1 mixture of lanolin and vaseline therapy for 7 and 14 days, respectively. One percent statin plus 1:1 mixture of lanolin and vaseline was used in the fifth and sixth groups for 7 and 14 days, respectively; and in seventh and eighth groups, 5% statin plus 1:1 mixture of lanolin and vaseline therapy was used for 7 and 14 days, respectively. All wounds were cleaned daily with sterile normal saline solution. After cleaning, cream base and statin creams were applied to the wounds. All creams were applied evenly in sufficient amounts to cover all wound areas. Evaluation of Healing After appropriate time points after the treatment started (7 and 14 days, 7 animals from each groups), the animals were euthanized by ether inhalation, and the tissue, including the wound and its surrounding skin and muscle, was excised. Wound healing was examined by measuring the reduction of the wound surface area with a stereomicroscope (SZX-ILLD2200, Olympus, Center Valley, PA) and photographed (Spot Insight QE; Diagnostic Instruments, Sterling Heights, MI). The ratio of healing was calculated by the following equation: Healing ratio (%) ⫽ 100 ⫻ (1 ⫺ wound area/initial wound area) Histologic Evaluations After macroscopic evaluation, the excised tissue was fixed with 10% buffered formalin. Each specimen was embedded in a paraffin block, sliced into thin 2.5-␮m sections, and stained with hematoxylin-eosin. Then, the specimens were assessed for severity of histopathologic changes, under light microscopy, by a pathologist who was unaware of the experimental procedure. Epidermal and dermal regeneration, granulation tissue thickness, and angiogenesis were considered as criteria for healing.18 Statistical Analyses Statistical differences between several treatments and their respective control were determined by Mann–Whitney U test, using SPSS 14.0 software. The level of significance was set at P ⬍ 0.05.

FIGURE 1. (A) The view of the wound in diabetic control group on the 14th day. (B) The view of the wound in lanolinvaseline group on the 14th day. (C) The view of the wound in 14 days 1% statin group. (D) The view of the wound in 14 days 5% statin group.

According to the statistical analyses, 1% statin 7 days group’s wound healing was better when compared with those of control-7 days group, and the results were statistically significant. Similarly, we found significant results in the comparison of 5% statin-7 days group and control-7 days group, control-14days [Figure 1(A)] and 1% statin-14 days [Figure 1(C)], control-14 days and 5% statin-14 days [Figure 1(D)], lanelin-vaseline-7 days and 1% statin-7 days, lanoline-vaseline-7 days and 5% statin-7 days, 1% statin-7 days and 1% statin-14 days, 5% statin-7 days and 5% statin-14 days, lanoline-vaseline-14 days and 1% statin-14 days, and lanolinevaseline-14 days [Figure 1(B)] and 5% statin-14 days groups (P ⬍ 0.05). It is understood that the efficacy of the drug is much more related with the treatment time than with the dosage because we could not find significant differences in wound healing in groups between 1% statin-7 days and 5% statin-7 days, 1% statin-14 days and 5% statin-14 days (P ⬎ 0.05). Furthermore, the differences were not statistically significant between the groups of control-14 days and lanoline-vaseline-14 days, and control-7 days and lanoline-vaseline-7 days. In the statistical analyses of the mean histopathologic scores and angiogenesis scores, we found statistically significant difference between the groups of 1% statin-14 days (Figure 2) and control-14 days, 5% statin-14 days and control-14 days, 1% statin 7 and 14 days, and 5% statin 7 and 14 days, which were also supporting the conclusion about the efficacy of treatment time and inefficacy of drug percentage. We could not find significant difference in histopathologic and angiogenesis scores between 7-days 1% statin and control groups (Figure 2), 7-days 5% statin and control groups, control-7 and 14 days, 7-days 1% and 5% statin groups, and 14-days 1% and 5% statin groups.

RESULTS Accordingly, in the multiple comparisons, the rates of wound healing were found to be significantly higher in the diabetic rat groups administered 1% and 5% atorvastatin compared with those administered a mixture of lanolin-vaseline and the untreated group (for comparison each one P ⬍ 0.05).

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DISCUSSION Chronic, nonhealing wounds are a significant medical problem among persons with DM and in the elderly population. Healthcare costs for a single ulcer event have been estimated at nearly $5000 to as much as $28,000 for all care up to 2 years after diagnosis.19 Volume 338, Number 3, September 2009

Effect of Statins on Diabetic Wounds

FIGURE 2. (A) A fresh wound site showed evident poorly formed granulation tissue, with altered epidermal and dermal organization. (B) A good re-epithelialization and well-formed granulation tissue is shown in a sample from 14 days 1% statin group.

The outcome of management of diabetic foot ulcers is poor and there is uncertainty concerning optimal approaches to management.2 The healing process requires a sophisticated interaction among inflammatory cells, biochemical mediators, extracellular matrix molecules, and microenvironmental cell population. All these events are stimulated by a number of mitogens and chemotactic factors.18 The basic biological and molecular events after cutaneous injury, with information mainly derived by studying experimental wounds in animals, cannot be separated and catogorized in a clear-cut way,20 however, it is clear that chronic nonhealing wounds do not follow the way of classical healing process. Hemodynamic abnormalities, hypoperfusion, and neuronal ischemia have been shown to be important pathogenic factors of diabetic neuropathy.21 In studies performed on diabetic animals, vasodilator agents (eg, angiotensin-converting enzyme inhibitors, a1-antagonists) and experimental agents (eg, aldose reductase inhibitors, protein kinase C inhibitors) have been shown to be able to improve neural perfusion.22 If diabetic neuropathy and ischemia are thought to play an important role in development of diabetic foot ulcers, one may surmise that the use of statins can be useful.23 Statins, widely used for the treatment of hyperlipidemia, have been shown to prevent cardiovascular events in patients with diabetes. In addition to preventing macrovascular diseases, statins may also be able to retard the progression of microvascular complications of diabetes.24 Statin drugs directly enhance the ability of endothelial nitric oxide synthase to generate nitric oxide in endothelial cells independent of lipid-lowering effects.25,26 In animal studies, the use of statins on the vascular system, such as the coronary artery, cerebral artery, small mesenteric artery, aorta, and corpus cavernosum, was shown to result in vascular relaxation by upregulating nitric oxide synthase.26 –28 Indeed, in addition to reducing lipid levels, these agents can improve endothelial function and reduce oxidative stress, which can improve microvascular © 2009 Lippincott Williams & Wilkins

function. In addition, statins cause a downregulation of the preproendothelin-1 mRNA level in endothelial cells, and thus reduce the synthesis of endothelin-1,29 which is a powerful vasoconstrictor. Intensive lipid-lowering treatment has been shown to be beneficial by reducing vascular response to angiotensin-2 (AT-2) and to increase endothelium-dependent vasodilatation.30 It was indicated that AT-2 induces T-type Ca2⫹ channels in endothelial cells, which may play a role in the development of vascular disorders, and AT-2–induced expression of alpha 1G was inhibited by treatment with atorvastatin.31 In addition, statins interfere with platelet aggregation and promote stabilization of atherosclerotic plaques. In vitro and in vivo studies have shown that statins exert strong antithrombotic effect.32,33 Statins have also been demonstrated to promote the neovascularization of ischemic tissue in normocholesterolemic animals by further increasing the functional activity of endothelial progenitor cells.34 Because ischemia may play an important role in the development of diabetic foot ulcers, a better understanding of the importance of oxygenation and tissue perfusion in lower extremities is essential. However, diabetic angiopathy, in both microlevel (such as vasa nervorum) and macrolevel (small-middle diameter arteries and arterioles), can result in an impairment of tissue functions by leading to ischemia.35,36 Statins, in this case, may be effective for the prevention of development of DFU as well as for the treatment of DFU via increased oxygenation and nourishment of tissues by increasing perfusion by the mechanisms mentioned earlier.15 In addition, growth factors in the form of gels have been shown to promote wound healing in diabetic foot ulcers.23 In our study, we found significantly higher scores not only for wound healing ratio macroscopically and mean histopathologic scores but also for scores of angiogenesis, especially in 14-day groups. This may reveal that 7 days for statin treatment can provide a significant benefit in macroscopic healing, but a longer statin treatment is needed for angiogenesis and other microscopic changes and this may be 14 days. As a conclusion, we state that statins have beneficial effects when used on diabetic wounds and may be used as a topical agent. Therefore, this study may provide an innovation in diabetic wound treatment. We also point out that healing with 1% statin was just as good as with 5% statin. REFERENCES 1. Valls MD, Cronstein BN, Montesinos MC. Adenosine receptor agonists for promotion of dermal wound healing. Biochem Pharmacol 2009;77:1177–24. 2. Hinchliffe RJ, Valk GD, Apelqvist J, et al. A systematic review of the effectiveness of interventions to enhance the healing of chronic ulcers of the foot in diabetes. Diabetes Metab Res Rev 2008;24(suppl 1): S119 –S144. 3. Gary Sibbald R, Woo KY. The biology of chronic foot ulcers in persons with diabetes. Diabetes Metab Res Rev 2008;24(suppl 1):S25–S30. 4. Richard JL, Schuldiner S. [Epidemiology of diabetic foot problems]. Rev Med Interne 2008;29(suppl 2):S222–S230. 5. Lavery L, Armstrong D, Wunderlich R, et al. Diabetic foot syndrome: evaluating the prevalence and incidence of foot pathology in Mexican Americans and non-Hispanic whites from a diabetes disease management cohort. Diabetes Care 2003;26:1435. 6. Reiber G. The epidemiology of diabetic foot problems. Diabetes Med 1996;13:S6. 7. Trousdale RK, Jacobs S, Simhaee DA, et al. Wound closure and metabolic parameter variability in a db/db mouse model for diabetic ulcers. J Surg Res 2009;151:100 –7. 8. Shukrimi A, Sulaiman AR, Halim AY, et al. A comparative study

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