Statins for Oxidative Stress and Mitochondrial Function in Diabetic Polyneuropathy
Condition: Oxidative Stress; Diabetic Polyneuropathy
MeSH terms: Polyneuropathies; Diabetic Neuropathies
Study type: Interventional
Phase: Phase 2

Summary: 
      Aims: To evaluate the effect of ezetimibe/simvastatin and rosuvastatin on oxidative stress
      and mitochondrial function in patients with DPN.

      Methods: We performed a randomized, double-blinded, placebo-controlled phase II clinical
      trial in adult patients with type 2 Diabetes Mellitus (T2DM) who had Diabetic Polyneuropathy
      (DPN) evaluated by composite scores and nerve conduction studies (NCS), HBA1C <12% (108
      mmol/mol), previous exclusion of other neuropathies. Ninety-eight persons with T2DM were
      allocated 1:1:1 to either placebo, ezetimibe/simvastatin 10/20 mg or rosuvastatin 20 mg for
      16 weeks, and healthy controls (not randomized) were included for comparisons. Primary
      outcomes were lipid peroxidation (LPO), nitric oxide (NO), and total antioxidant capacity
      (TAC); secondary were clinical, NCS and metabolic parameters. Results were expressed as mean
      ± standard deviation (SD) or standard error of the mean (SEM), frequencies and percentages.
      Non-parametric analysis was used.
    

Detailed description: 
      Introduction Nerve dysfunction system in patients with diabetes is known as diabetic
      neuropathy and is considered as the most prevalent microvascular complication -up to 60%- in
      type 2 Diabetes Mellitus (T2DM) subjects. Diabetic polyneuropathy (DPN) comprise ≈70% of all
      cases. Its diagnosis is established by means of validated scores based on clinical features
      and abnormal nerve conduction studies (NCS). Pathophysiologic findings include loss of
      multifocal and focal nerve fibers secondary to axonal degeneration an segmental
      demyelinization, basically due to oxidative stress induced by chronic hyperglycemia, which
      leads to neural apoptosis. Other mechanisms involved in peripheral nerve injure is
      nitrosative stress induced by nitric oxide (NO).

      Ezetimibe diminishes cholesterol esters content in chylomicrons by reducing liver cholesterol
      intake which in consequence increases LDL uptake and plasma depuration; as monotherapy it
      reduces LDL-C by 17%. When combined with simvastatin, cholesterol reduction is potentially
      increased; furthermore, pleiotropic effects of statins include an increase on nuclear factor
      kappa B activity and amelioration of superoxide ions after 12 week treatment. Another
      hydroxy-methyl-glutaryl coenzyme A inhibitor, rosuvastatin, has an antioxidant effect by
      acting as free radical carrier diminishing mitochondrial and cellular lipid peroxidation
      (LPO) production.

      We conducted this study to evaluate the effect of ezetimibe/simvastatin and rosuvastatin on
      oxidative stress in patients with DPN.

      Methods Study design A randomized, double blinded, placebo controlled phase II clinical trial
      was performed in the Clinic and Experimental Therapeutics Institute, University of
      Guadalajara, Mexico. Subjects were assigned to three group treatments in blocks with a
      parallel sequence 1:1:1 through a randomized computer-based list, generated by a different
      researcher unaware of drugs given. Patients were divided to: control group that received
      placebo, ezetimibe/simvastatin and rosuvastatin as a daily single dose for 16 weeks. The
      selection period was performed from February 2012 to January 2013. We selected 5
      non-randomized healthy subjects (HS) from a blood bank to compare the oxidative stress
      status.

      Study population Inclusion criteria were ≥18 years old, T2DM according to American Diabetes
      Association and DPN by Dyck et. al.3 criteria, HbA1c <12%, and informed consent signed. They
      were excluded if renal or hepatic failure, pregnant or breastfeeding, other neuropathies
      (alcohol-induced, radiculopathy, autoimmune, cancer-related), and eliminated if lack
      treatment adherence (<80% of drug intake), severe adverse drug reaction and/or serious health
      illness. Patients were selected by invitation in forums; outpatients recruited from primary
      care clinics; and database collected previously by our Institute from February 2010 to 2012.
      Patients were instructed to take their drugs only by night at the same time every day, as
      follows: placebo 100 mg, ezetimibe/simvastatin 10/20 mg, and rosuvastatin 20 mg. All drugs
      were similar in physical characteristics and presented in dark vials, carefully filled by
      another group researcher, who placed a respective tag with the patient code. Also, patients
      were provided with a diary where they would write down the date and time of drug
      administration, and drug adverse reactions felt. Such information was collected and
      registered every 4 weeks. Primary outcomes were oxidative stress markers LPO, NO, and TAC
      before and after 16 week intervention. Secondary outcomes were clinical, NCS and metabolic
      [fasting glucose, HbA1c, total cholesterol, high and low density lipoproteins (HDL, LDL), and
      triglycerides] parameters. Safety profile was assessed with drug adverse reactions, renal
      (urea, creatinin), and hepatic [(alamin- and aspartate-aminotransferase,
      gama-glutamyltransferase, bilirubins and phosphokinase] laboratory variables.

      Oxidative stress and mitochondrial function markers LPO was measured according to kit
      specifications (Oxford Biomedical Research Inc., FR12), 200 μL of serum where processed with
      a chromogen substance that reacts with malondialdehyde (MDA) and 4-hydroxy-alkenals (HNA),
      the absorbance measured at 586 nm, and results expressed in nmol/mL.

      Previous deproteinization of the samples, we performed a colorimetric for determining the
      concentration of NO with 85 µL of serum (Nitric Oxide Assay Kit, User protocol 482650), with
      results expressed as pmol/mL.

      Total antioxidant capacity (TAC) was realized with 200 µL of serum, to obtain values of
      millimole (mM) equivalent of uric acid (Total Antioxidant Power Kit, No. 02090130, Oxford
      Biomedical Research®).

      Clinical and nerve conduction variables Neuropathic symptoms (NSS) and disability scores
      (NIS) described by Dyck, et. al. were obtained by physical examination and anamnesis. We also
      measured the latency, duration, amplitude and motor nerve conduction velocity from fibula,
      tibial, median and ulnae nerves, and sensitivity parameters from sural, median and ulnae
      nerves, as required by the American Association of Electrodiagnostic Medicine.

      Ethical considerations The study was approved by the Research and Ethics Committee of the
      Health Science University Center, University of Guadalajara, Mexico. Identification codes
      were assigned to each participant to guarantee patient confidentiality, and an informed
      consent form was signed before entering the protocol, according to national and international
      laws, and also as stipulated by the Helsinki Statements (http://www.wma.net/es/30
      publications/ 10 policies/b3/17c.pdf, accessed January 2011).

      Statistical analysis The sample size was obtained by a clinical study design formula taking
      in account a difference change of 0.05 nmol/mL in LPO, 95% confidence interval, 80% potency,
      and two-tailed p<0.05, which resulted in 21 for each group. Quantitative variables were
      expressed as mean ± standard deviation. Kolmogorov-Smirnov and Shapiro-Wilk tests were
      performed to determine the non-parametric distribution of variables. Friedman and Wilcoxon
      tests were realized for before and after measurements, and Kruskal-Wallis with Mann-Whitney´s
      U as post-hoc analysis for between group comparisons. Qualitative variables were expressed as
      frequencies and percentages. Chi square test was used to evaluate differences in dichotomy
      variables before and after treatment, between group comparisons were determined by Fisher´s
      exact test and χ2 as needed. Significance level was established with p value <0.05.
    

Intervention name(s): calcined magnesia; Ezetimibe/simvastatin; Rosuvastatin
Intervention type(s): Drug; Drug; Drug
Intervention description(s): We ensured the patient took the drug at night before meals; We ensured the patient took the drug at night before meals; We ensured the patient took the drug at night before meals
Other intervention name(s): placebo; Zintrepid; Vytorin; Crestor
Intervention MeSH terms: Magnesium Oxide; Rosuvastatin Calcium; Simvastatin; Ezetimibe; Ezetimibe, Simvastatin Drug Combination
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Treatment
Keywords: ezetimibe/simvastatin; rosuvastatin; diabetic polyneuropathy; oxidative stress; mitochondrial dysfunction