Neurol Sci DOI 10.1007/s10072-014-1647-2
ORIGINAL ARTICLE
Minocycline improves peripheral and autonomic neuropathy in type 2 diabetes: MIND study Ashit Syngle • Inderjeet Verma • Pawan Krishan Nidhi Garg • Vijaita Syngle
•
Received: 22 September 2013 / Accepted: 17 January 2014 Ó Springer-Verlag Italia 2014
Abstract Diabetic peripheral neuropathy and diabetic autonomic neuropathy are serious and common complications of diabetes associated with increased risk of mortality and cardiovascular disease. We sought to evaluate the safety and efficacy of minocycline in type 2 diabetic patients with diabetic peripheral and autonomic neuropathy. In a randomized placebo controlled study, 50 outpatients were randomly assigned to receive 100 mg minocycline or placebo. Outcome measures included the vibration perception threshold (VPT), Leeds assessment of neuropathic symptoms and signs (LANSS), Pain Disability Index (PDI), Visual Analog Scale (VAS), beck depression inventory (BDI), health assessment questionnaire (HAQ) and autonomic neuropathy, assessed by cardiovascular reflex tests according to Ewing and peripheral sympathetic autonomic function was assessed by FDA approved Sudoscan. At baseline there were no significant differences between demographic variables and the neuropathy variables in the minocycline and placebo groups. After treatment, VPT significantly improved in the minocycline group as compared to the placebo group. Mean posttreatment scores on the LANSS, PDI and HAQ were significantly lower in the minocycline group compared with the A. Syngle (&) Healing Touch City Clinic, Chandigarh and Senior Consultant Physician and Rheumatologist Fortis Multi Specialty Hospital, Mohali, India e-mail: [email protected] I. Verma P. Krishan N. Garg Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India V. Syngle Healing Touch City Clinic, House No. 547, Sector 16-D, Chandigarh 160036, India
placebo group. However, BDI and VAS significantly (p = 0.01) improved in both minocycline and placebo groups (Table 2). After treatment with minocycline, heart rate (HR) response to standing significantly improved, while there was a borderline significance toward a reduction in HR response to deep breath. These finding indicate that 6-week oral treatment with minocycline is safe, well tolerated and significantly improves peripheral and autonomic neuropathy in type 2 diabetic patients. Keywords Minocycline Diabetic neuropathy Cardiovascular autonomic neuropathy Vibration perception threshold Leeds assessment of neuropathic symptoms and signs Sudoscan
Introduction Diabetic neuropathy (DN) is a serious and common complication of diabetes often associated with increased risk of mortality and cardiovascular disease [1] and it appears relatively early in the disease process. Globally, diabetic neuropathy affects approximately 1.9 % of the population. Progression of diabetic neuropathy causes various problems in daily life and affects the prognosis of diabetic patients [1]. The DN affecting different parts of the nervous system presents with diverse clinical manifestations. Most common among the neuropathies are diabetic peripheral neuropathy (DPN) and the diabetic autonomic neuropathy (DAN) [2]. DPN may be asymptomatic and is the leading cause of foot amputation. DPN affects up to 60–70 % of diabetic patients [3]. Cardiovascular autonomic neuropathy (CAN) is the most studied and clinically important form of diabetic autonomic neuropathy [4]. DAN may affect many organ systems
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throughout the body (e.g. gastrointestinal, heart, lungs and blood vessels and genitourinary). 20 % of asymptomatic diabetic patients have CAN [4]. Several trials have shown that strict glycemic control reduces the occurrence and progression of diabetes-related neuropathy [5, 6]. However, this approach alone does not completely eliminate complications of diabetes like peripheral and autonomic neuropathy (AN). Thus, the development of new drug molecule for early recognition to manage DN remains a high priority. Minocycline has been tested in streptozotocin-induced DN in Wistar rats. Three weeks of treatment with a combination of minocycline and aspirin showed significant improvement in peripheral neuropathy [7]. Minocycline is a second-generation, broad spectrum tetracycline, which is widely used as an antibiotic since 1970s. Minocycline is readily available, inexpensive and relatively safe when administered long term. It has been known for some time that minocycline has protective properties and beneficial effects on the chronic complications of diabetes, including blocking matrix metalloproteinase, treating retinopathy, preventing death of specialized kidney cells, and inhibiting microglia activation and proliferation [8–11]. While laboratory evidence supports minocycline as a potential treatment for diabetic complications, little is known about its efficacy in humans. Hence, we decided to investigate its safety and efficacy in diabetic peripheral and autonomic neuropathy in type 2 diabetes.
a full explanation of the study. Inclusion criteria were (1) type 2 diabetes according to the World Health Organization; (2) stable diabetic control as evidenced by Hemoglobin A1c (HbA1c) level B11 % at baseline; (3) history of neuropathic pain between 6 months and 5 years in duration; (4) pain rating of C50 units on the Visual Analog Scale (VAS) at the first screening visit and impaired vibration perception threshold (VPT) of C25 V. Exclusion criteria were (1) neuropathy pain and significant neurological disorders other than that of diabetic origin; (2) pregnant or lactating mothers; (3) patients with the history of peptic ulcers, SLE, liver disease, pulmonary tuberculosis, thyroid disorders, uremia, vitamin deficiency, hereditary, paraneoplastic neuropathy, alcoholism, smokers and patients on steroid therapy, neuroprotective drugs, and any medication that may adversely influence autonomic function were excluded; (4) hypersensitivity to minocycline; (5) neutropenia, anemia, or thrombocytopenia; (6) participation in a study of any investigational drug within 3 months before the study; (7) clinically significant hepatic dysfunction (liver function tests [2 times the upper limits), and renal insufficiency (creatinine clearance \30 mL/min). Patients were randomly assigned to be treated with minocycline (100 mg) or placebo two times a day in addition to their existing treatment. The study patients were not prescribed any other approved treatment for neuropathy apart from study medication/placebo. However, acetaminophen (500 mg tablets) as rescue medication was allowed at maximum dose of 4 g/day.
Study design/methods
Assessment of peripheral neuropathy
Study design
All patients underwent a detailed clinical examination to rule out any other cause of neuropathy. Vibration perception threshold was measured by biothesiometer (BioMedical Instrument Company, Newbury, Ohio, United States) approved by FDA. [12]. The Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) Pain Scale is an assessment tool used by the medical community to analyze and classify pain. The primary purpose of this test is to assess whether the pain experienced is predominantly due to nerve damage or not [13]. The Pain Disability Index (PDI) a simple and rapid instrument for measuring the impact that pain has on the ability of a person to participate in essential life activities [14]. A VAS is a measurement instrument that tries to measure a characteristic or attitude that is believed to range across a continuum of values and cannot easily be directly measured. The Beck Depression Inventory (BDI) is one of the most widely used instruments for measuring the severity of depression [15].
A prospective, open label, randomized placebo controlled pilot study of 6 weeks duration was carried out in a single center with the same investigators doing all assessments. Study designed to determine the usefulness of minocycline for DPN and DAN in diabetic patients was reviewed and approved by the Institutional Ethics Committee of Punjabi University Patiala, India. The investigator involved in assessment of peripheral neuropathy, CAN and Sudoscan were blinded to the treatment protocol. All variables were assessed at baseline and 6 weeks. Selection of patients Patients with type 2 diabetes who were C18 years of age were eligible to participate in the research study. The study was performed in accordance with the declaration of Helsinki and the code of Good Clinical Practice. All patients were provided written informed consent to participate after
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Assessment of autonomic neuropathy A battery of non-invasive tests was used for accurate assessment of AN function which was based on assessment of cardiovascular reflex tests diagnosed according to Ewing [16] and peripheral sympathetic autonomic function assessed by FDA approved Sudoscan (Impeto Medical Device, EZS 01750010193, Paris, France) [17]. Symptoms of ANS were assessed by administrating the survey of autonomic symptoms [18]. Health assessment based on daily activities was measured using Health Assessment Questionnaire (HAQ). HAQ connected with ‘quality of life’ and ‘disability’ [19]. Laboratory analysis Glycosylated hemoglobin (HbA1c) was determined at baseline and after 6 weeks of oral treatment. Safety parameters were determined at baseline and 6 weeks of oral treatment. They included hemoglobin, full blood count, liver enzymes, total protein, bilirubin, creatinine, uric acid, cholesterol, triglycerides, vitamin B12 and urine microalbuminuria. Statistical analysis Test values are reported as mean ± SEM. The Student’s t test was used to compare the continuous variables of autonomic neuropathy for treatment and placebo group. Multivariate correlation analysis was used to find the correlation between the neuropathy variables, biochemical parameters and diseases activity. A p value \0.05 were considered statistically significant. Statistical analysis was done using the Prism Graph Pad program for Windows 7.0.
Results A total of 50 type 2 diabetes patients eligible for the study gave informed consent to participate in the research study. The treatment group had 25 patients with mean age 58.50 ± 2.0 (9 females and 16 males) compared with 25 in the placebo group with mean age 60.46 ± 0.95 (7 females and 18 males). The demographic and laboratory features of treatment group and placebo group are summarized in Table 1. We found that there was no significant difference in the demographic and laboratory parameters of minocycline group and placebo group. The posttreatment changes in the neurological, CAN variables and sudomotor functions are shown in Table 2. Mean posttreatment score of VPT significantly lower in the minocycline group compared with the placebo group (22.29 ± 1.6 vs. 36.67. 1.6 ± 1.9, p = 0.01 vs. p = 0.80) (Table 2).
Table 1 The demographic and laboratory features of treatment and placebo group Minocycline (n = 25)
Placebo (n = 25)
p value
Age (years)
58.50 ± 2.0
60.46 ± 0.95
0.38
Sex (F/M)
9/16
7/18
–
Disease duration (years) BMI (kg/m2)
13.50 ± 1.48 26.29 ± 0.69
12.35 ± 0.84 25.17 ± 0.92
0.50 0.14 0.63
HbA1c (%)
7.15 ± 0.13
7.25 ± 0.15
Fasting sugar (mg/dL)
151.9 ± 13.31
159.6 ± 10.87
0.60
Systolic blood pressure (mm Hg)
131.8 ± 1.90
134.3 ± 1.68
0.34
Diastolic blood pressure (mm Hg)
82.08 ± 1.36
83.67 ± 1.36
0.41
Hb (g/Dl)
12.29 ± 0.29
12.36 ± 0.31
0.86
Uric acid (mg/dL) Serum creatinine (mg %)
5.24 ± 0.18 1.05 ± 0.04
5.02 ± 0.12 0.92 ± 0.02
0.33 0.02
Vitamin B12 (pg/mL)
635.8 ± 61.6
591.3 ± 53.4
0.58
Urine microalbuminuria
17.48 ± 0.99
15.87 ± 0.73
0.20
Data are means ± SEM. p \ 0.05 considered significantly F female, M male, BMI Body Mass Index
34.4 % of study patients treated with 6 weeks of minocycline improved while there was worsening of peripheral neuropathy in 0.06 % of patients in the placebo arm. LANSS and PDI were significantly (p B 0.01) improved in the minocycline group as compared to the placebo group (Table 2). However, BDI and VAS were significantly (p = 0.01) improved in both minocycline and placebo groups (Table 2). But the percentage improvement was higher in the minocycline group than in the placebo when improvement was measured on the BDI (53.98 vs. 23.94 %), and VAS (56.12 vs. 17.32 %) (Table 2). Heart rate (HR) response to standing significantly (p = 0.01) improved after treatment with minocycline (Table 2). After completion of the oral treatment period, there was a borderline significance toward a reduction in HR response to deep breath only in the minocycline-treated group as compared to the placebo-treated patients (Table 2). Other variables of CAN were not significantly improved in both minocycline- and placebo-treated group. Sudomotor function did not improve significantly in either minocycline-(p = 0.13) or placebo (p = 0.22)-treated diabetic patients (Table 2). After treatment with minocycline the quality of life assessed by HAQ was significantly improved in treatment group (62.74 %, p = 0.01) as compared to the placebo (25.45 %, p = 0.12) group (Table 2). There were a few minor number side effects in the minocycline group which included dizziness, nausea and vomiting. One patient complained of rashes following sun exposure.
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Neurol Sci Table 2 Effect of minocycline and placebo after 6 weeks of treatment on neurological variables, CAN, Sudoscan and HAQ in type 2 diabetic patients Variables
Minocycline 0 week
Group 6 week
p value
Placebo 0 week
Group 6 week
p value
34.00 ± 2.23
22.29 ± 1.65
\0.001
36.44 ± 1.94
36.67 ± 1.69
0.80
12.9 ± 1.12 14.8 ± 1.2
7.34 ± 0.12 8.3 ± 0.60
0.01 0.001
12.1 ± 0.02 14.13 ± 1.7
10.2 ± 1.23 11.92 ± 0.35
0.09 0.07
Neurological VPT LAANS PDI VAS
6.7 ± 0.11
2.9 ± 0.44
0.01
5.6 ± 0.22
4.63 ± 0.08
0.01
BDI
23.19 ± 0.35
10.67 ± 0.31
0.01
20.04 ± 0.54
14.96 ± 0.63
0.01
CAN variables HRV
1.26 ± 0.02
1.29 ± 0.04
0.16
1.22 ± 0.02
1.20 ± 0.03
0.14
HRD
9.54 ± 1.24
11.08 ± 1.04
0.06
10.08 ± 0.99
10.42 ± 0.90
0.45
HRS
1.00 ± 0.01
1.04 ± 0.06
0.01
0.97 ± 0.02
0.97 ± 0.02
0.08
BPS
7.75 ± 1.02
7.25 ± 0.89
0.37
8.58 ± 0.98
8.58 ± 0.98
0.90
BPH
14.25 ± 1.05
14.96 ± 5.15
0.45
13.13 ± 1.0
13.13 ± 1.06
0.78
Sudoscan
54.77 ± 3.96
59.34 ± 4.06
0.13
59.40 ± 2.7
59. 40 ± 2.76
0.22
1.9 ± 0.12
0.01
5.5 ± 1.3
HAQ
5.1 ± 1.2
4.1 ± 1.2
0.12
Data are means ± SEM. p \ 0.05 considered significantly VPT vibration perception threshold, LAANS Leeds Assessment of Neuropathic Symptoms and Signs, PDI Pain Disability Index, VAS Visual Analog Scale, BDI beck depression inventor, HRV HR response to valsalva, HRD HR response to deep breath, HRS HR response to standing, BPS BP response to standing, BPH BP response to handgrip, HAQ health assessment questionnaire
Changes in glycemic control of the patients and placebo group were followed for 6 weeks of treatment. In the control group, HbA1c (mean ± SEM) at baseline and after treatment was 7.25 ± 0.15 and 7.07 ± 0.16 % (p = 0.04), respectively. Corresponding values in the minocycline group was 7.15 ± 0.13 and 6.56 ± 0.25 % (p = 0.02), respectively. There were no significant differences between the two groups at baseline (Table 1). There was statistically significant correlation between VPT and HbA1c before (p = 0.02) and after (p = 0.01) minocycline therapy. VPT significantly correlated with BDI (p = 0.02), VAS (p = 0.03) heart rate response to deep breath (p = 0.004) and standing (p = 0.007) and Sudoscan (p = 0.001) after minocycline treatment. There was no statistically significant correlation among VPT and HbA1c, BDI, PDI, VAS, LANSS and autonomic neuropathy variables in placebo group.
Discussion Diabetic peripheral neuropathy and autonomic neuropathy are the most common neuropathies occurring in diabetes patients [1]. The early diagnosis and appropriate treatment of diabetic neuropathy is important because of patients being at higher risk of insensate injury to their feet due to DPN and also higher risk of morbidity and increased mortality, particularly if CAN is present [2]. As yet, there is no effective treatment to prevent the development and
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progression of human diabetic neuropathy. Therapeutic interventions that have been assessed include optimal glycemic control, aldose reductase inhibitors, angiotensin converting enzyme inhibitors, antioxidants (gamma linoleic acid, alpha lipoic acid, vitamin E) amino guanidine, advanced glycation end product inhibitor, C-peptide and Protein kinase C b inhibitor (which increase nerve blood flow) [2]. Some of these interventions have been withdrawn due to side effects and others are ineffective. Currently, duloxetine and pregabalin (both FDA approved) are the only medication that have been used for the treatment of peripheral neuropathic pain in diabetes [20]. The efficacies of minocycline on peripheral and autonomic neuropathy have not been previously investigated in type 2 diabetes while laboratory evidence supports its potential role in treatment of diabetic complications. An experimental animal study has reported a significant improvement in diabetic neuropathy after 3 weeks of treatment with minocycline in streptozotocin-induced diabetic neuropathy rats [7]. The results of the present study demonstrate that minocycline significantly improved VPT and painful diabetic neuropathy, i.e. LANSS and albeit small, improvement in HR response to standing and borderline significance toward HR response to deep breath. LANSS is an extremely good instrument to assess whether pain experienced is predominantly due to nerve damage or not. It is also the only published tool with validity for discriminating between neuropathic and nociceptive pain, regardless of the disease-based diagnostic methods [13].
Neurol Sci
The improvement in peripheral and autonomic neuropathy shown in this study over a very short period is more impressive than the improvement shown in trials of angiotensin converting enzyme inhibitor, aldose reductase inhibitors, alpha lipoic acid (ALADIN AND DEKAN study) and vitamin E [21–26], since minocycline has demonstrated a potential to improve both peripheral and autonomic neuropathies. The exact mechanisms by which minocycline exerted its effect on peripheral neuropathy are not known, but it could be related to its property to inhibit the enhanced production of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and reduction in glycemic control. The over production of these MMPs is responsible for the degradation or remodeling of the extracellular matrix (ECM) [26] and in addition, MMP-9 induces neuropathic pain through interleukin (IL)-1b cleavage and microglia activation, following nerve injury [27, 28]. This leads to damage of the vascular bed supplying nutritive blood to the sensory nerve fibers and responsible for peripheral nerve dysfunction in diabetes [28]. In the present study, we also observed that minocycline improved autonomic function as compared to the placebo group. There is recent evidence of activation of inflammatory cytokines IL-1 and IL-6 due to systemic inflammation [29]. Activation of these inflammatory cytokine significantly correlates with abnormalities in sympathetic vagal balance [29]. Minocycline possibly exerts its antiinflammatory effects through inhibition of inflammatory cytokines like IL-6, IL-1b, TNF-a and IL-8 [30–32] and reduction in mitogen-activated protein kinase (MAPK) pathways in microglial activation and proliferation [33]. Microglia are the primary immune effector cells of the central nervous system and play an important role in the initiation of inflammatory responses through inflammatory cytokines and chemokines [34, 35]. In this study, glycemic status improved in both the treatment and placebo groups, but improvement in minocycline group is much more than the placebo group. This is possibly due to inhibition of MMP-9 by minocycline [27]. In a recent study, HbA1c has been shown to be significantly associated with MMP-9 concentration in type 2 diabetes [36]. Hence, minocycline has a dual benefit in diabetic neuropathy. In this study, minocycline significantly improved LAANS, PDI score and VAS for pain. The improvement in LAANS, PDI score and VAS score suggests that minocycline could have the ability to improve neuropathic pain and improvement of glycemic status through inhibition of MMP-9 [27, 28]. Neuropathic pain is highly problematic for diabetic patients because it is often worse at night, depriving patients of sleep and thereby causing fatigue. In addition, it also has a strong negative effect on quality of
life (QOL) [20]. Interestingly, in the present study QOL assessed by HAQ significantly improved in minocycline group but not in placebo group. Patient VAS for pain also significantly improved in placebo group. The improvement in diabetic neuropathic pain in placebo group appears to be related to improvement in glycemic status since painful diabetic neuropathy is inversely related to the degree of glycemic control [37]. The percentage improvement in VAS is greater in minocycline group (56.12 %) as compared to placebo group (17.32 %) and also there was improvement in VPT, LAANS, PDI, QOL and HR response to standing in the study drug group only and not in the placebo group. It would thus appear that improvement in glycemic control alone cannot account for these improvements. In our study, we have found positive correlation between VPT and HbA1c before and after treatment with minocycline. Previous research has shown a positive association between impaired VPT and high HbA1c in diabetes [38] and our study results are also consistent with these results suggesting that patients with hyperglycemia may have higher risk of peripheral neuropathy. VPT also correlated with BDI, VAS, HR response to deep breath, HR response to standing and sudomotor function after minocycline treatment suggesting that improvement of DPN after therapy with minocycline is associated with improvement in depression, pain and autonomic dysfunction. In this study only two patients reported minor side effects, i.e. nausea, vomiting, dizziness and patient reported photosensitivity. These did not necessitate drug discontinuation. It should be noted that no particularly severe events were observed, thus confirming the safety of minocycline for long-term administration. Furthermore, we observed that the patient compliance was very high in this study because of close follow-up and regular contact with patients and also because of short follow-up of the study. Strength of this study is that it is the first to translate laboratory evidence to human experience; study subjects were well-matched with good overall selection with good treatment compliance and follow-up. Side effects were minor and did not necessitate discontinuation of study drug. Apart from primary benefits, the study demonstrated several ancillary benefits. The study limitation is that it included a small number of patients for short duration, and peripheral neuropathy evaluation in this study mainly relied on neuropsychological testing and used VPT LANSS, VAS, PDI and BDI as physical measures while autonomic dysfunction was characterized by an extensive battery of tests. In conclusion, this study demonstrates that treatment with minocycline is safe and well tolerated and significantly improves peripheral neuropathy and autonomic neuropathy in type 2 diabetes. It is important to point out
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that these effects might have a positive influence on cardiac risk associated with diabetic neuropathy. In addition minocycline appear to exert a beneficial effect on the glycemic status. The results of this pilot study would surely further research in this direction. Acknowledgments We wish to thank to the people who agreed to participate as subjects in the research reported here. We also gratefully acknowledge the availability of Sudoscan in our center by Impeto Medical, Paris, France. We are very thankful to University Grant Commission, New Delhi, India for providing the research fellowship [No. F.10-15/2007 (SA-I)]. Conflict of interest
None.
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ORIGINAL ARTICLE
Minocycline improves peripheral and autonomic neuropathy in type 2 diabetes: MIND study Ashit Syngle • Inderjeet Verma • Pawan Krishan Nidhi Garg • Vijaita Syngle
•
Received: 22 September 2013 / Accepted: 17 January 2014 Ó Springer-Verlag Italia 2014
Abstract Diabetic peripheral neuropathy and diabetic autonomic neuropathy are serious and common complications of diabetes associated with increased risk of mortality and cardiovascular disease. We sought to evaluate the safety and efficacy of minocycline in type 2 diabetic patients with diabetic peripheral and autonomic neuropathy. In a randomized placebo controlled study, 50 outpatients were randomly assigned to receive 100 mg minocycline or placebo. Outcome measures included the vibration perception threshold (VPT), Leeds assessment of neuropathic symptoms and signs (LANSS), Pain Disability Index (PDI), Visual Analog Scale (VAS), beck depression inventory (BDI), health assessment questionnaire (HAQ) and autonomic neuropathy, assessed by cardiovascular reflex tests according to Ewing and peripheral sympathetic autonomic function was assessed by FDA approved Sudoscan. At baseline there were no significant differences between demographic variables and the neuropathy variables in the minocycline and placebo groups. After treatment, VPT significantly improved in the minocycline group as compared to the placebo group. Mean posttreatment scores on the LANSS, PDI and HAQ were significantly lower in the minocycline group compared with the A. Syngle (&) Healing Touch City Clinic, Chandigarh and Senior Consultant Physician and Rheumatologist Fortis Multi Specialty Hospital, Mohali, India e-mail: [email protected] I. Verma P. Krishan N. Garg Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India V. Syngle Healing Touch City Clinic, House No. 547, Sector 16-D, Chandigarh 160036, India
placebo group. However, BDI and VAS significantly (p = 0.01) improved in both minocycline and placebo groups (Table 2). After treatment with minocycline, heart rate (HR) response to standing significantly improved, while there was a borderline significance toward a reduction in HR response to deep breath. These finding indicate that 6-week oral treatment with minocycline is safe, well tolerated and significantly improves peripheral and autonomic neuropathy in type 2 diabetic patients. Keywords Minocycline Diabetic neuropathy Cardiovascular autonomic neuropathy Vibration perception threshold Leeds assessment of neuropathic symptoms and signs Sudoscan
Introduction Diabetic neuropathy (DN) is a serious and common complication of diabetes often associated with increased risk of mortality and cardiovascular disease [1] and it appears relatively early in the disease process. Globally, diabetic neuropathy affects approximately 1.9 % of the population. Progression of diabetic neuropathy causes various problems in daily life and affects the prognosis of diabetic patients [1]. The DN affecting different parts of the nervous system presents with diverse clinical manifestations. Most common among the neuropathies are diabetic peripheral neuropathy (DPN) and the diabetic autonomic neuropathy (DAN) [2]. DPN may be asymptomatic and is the leading cause of foot amputation. DPN affects up to 60–70 % of diabetic patients [3]. Cardiovascular autonomic neuropathy (CAN) is the most studied and clinically important form of diabetic autonomic neuropathy [4]. DAN may affect many organ systems
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throughout the body (e.g. gastrointestinal, heart, lungs and blood vessels and genitourinary). 20 % of asymptomatic diabetic patients have CAN [4]. Several trials have shown that strict glycemic control reduces the occurrence and progression of diabetes-related neuropathy [5, 6]. However, this approach alone does not completely eliminate complications of diabetes like peripheral and autonomic neuropathy (AN). Thus, the development of new drug molecule for early recognition to manage DN remains a high priority. Minocycline has been tested in streptozotocin-induced DN in Wistar rats. Three weeks of treatment with a combination of minocycline and aspirin showed significant improvement in peripheral neuropathy [7]. Minocycline is a second-generation, broad spectrum tetracycline, which is widely used as an antibiotic since 1970s. Minocycline is readily available, inexpensive and relatively safe when administered long term. It has been known for some time that minocycline has protective properties and beneficial effects on the chronic complications of diabetes, including blocking matrix metalloproteinase, treating retinopathy, preventing death of specialized kidney cells, and inhibiting microglia activation and proliferation [8–11]. While laboratory evidence supports minocycline as a potential treatment for diabetic complications, little is known about its efficacy in humans. Hence, we decided to investigate its safety and efficacy in diabetic peripheral and autonomic neuropathy in type 2 diabetes.
a full explanation of the study. Inclusion criteria were (1) type 2 diabetes according to the World Health Organization; (2) stable diabetic control as evidenced by Hemoglobin A1c (HbA1c) level B11 % at baseline; (3) history of neuropathic pain between 6 months and 5 years in duration; (4) pain rating of C50 units on the Visual Analog Scale (VAS) at the first screening visit and impaired vibration perception threshold (VPT) of C25 V. Exclusion criteria were (1) neuropathy pain and significant neurological disorders other than that of diabetic origin; (2) pregnant or lactating mothers; (3) patients with the history of peptic ulcers, SLE, liver disease, pulmonary tuberculosis, thyroid disorders, uremia, vitamin deficiency, hereditary, paraneoplastic neuropathy, alcoholism, smokers and patients on steroid therapy, neuroprotective drugs, and any medication that may adversely influence autonomic function were excluded; (4) hypersensitivity to minocycline; (5) neutropenia, anemia, or thrombocytopenia; (6) participation in a study of any investigational drug within 3 months before the study; (7) clinically significant hepatic dysfunction (liver function tests [2 times the upper limits), and renal insufficiency (creatinine clearance \30 mL/min). Patients were randomly assigned to be treated with minocycline (100 mg) or placebo two times a day in addition to their existing treatment. The study patients were not prescribed any other approved treatment for neuropathy apart from study medication/placebo. However, acetaminophen (500 mg tablets) as rescue medication was allowed at maximum dose of 4 g/day.
Study design/methods
Assessment of peripheral neuropathy
Study design
All patients underwent a detailed clinical examination to rule out any other cause of neuropathy. Vibration perception threshold was measured by biothesiometer (BioMedical Instrument Company, Newbury, Ohio, United States) approved by FDA. [12]. The Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) Pain Scale is an assessment tool used by the medical community to analyze and classify pain. The primary purpose of this test is to assess whether the pain experienced is predominantly due to nerve damage or not [13]. The Pain Disability Index (PDI) a simple and rapid instrument for measuring the impact that pain has on the ability of a person to participate in essential life activities [14]. A VAS is a measurement instrument that tries to measure a characteristic or attitude that is believed to range across a continuum of values and cannot easily be directly measured. The Beck Depression Inventory (BDI) is one of the most widely used instruments for measuring the severity of depression [15].
A prospective, open label, randomized placebo controlled pilot study of 6 weeks duration was carried out in a single center with the same investigators doing all assessments. Study designed to determine the usefulness of minocycline for DPN and DAN in diabetic patients was reviewed and approved by the Institutional Ethics Committee of Punjabi University Patiala, India. The investigator involved in assessment of peripheral neuropathy, CAN and Sudoscan were blinded to the treatment protocol. All variables were assessed at baseline and 6 weeks. Selection of patients Patients with type 2 diabetes who were C18 years of age were eligible to participate in the research study. The study was performed in accordance with the declaration of Helsinki and the code of Good Clinical Practice. All patients were provided written informed consent to participate after
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Assessment of autonomic neuropathy A battery of non-invasive tests was used for accurate assessment of AN function which was based on assessment of cardiovascular reflex tests diagnosed according to Ewing [16] and peripheral sympathetic autonomic function assessed by FDA approved Sudoscan (Impeto Medical Device, EZS 01750010193, Paris, France) [17]. Symptoms of ANS were assessed by administrating the survey of autonomic symptoms [18]. Health assessment based on daily activities was measured using Health Assessment Questionnaire (HAQ). HAQ connected with ‘quality of life’ and ‘disability’ [19]. Laboratory analysis Glycosylated hemoglobin (HbA1c) was determined at baseline and after 6 weeks of oral treatment. Safety parameters were determined at baseline and 6 weeks of oral treatment. They included hemoglobin, full blood count, liver enzymes, total protein, bilirubin, creatinine, uric acid, cholesterol, triglycerides, vitamin B12 and urine microalbuminuria. Statistical analysis Test values are reported as mean ± SEM. The Student’s t test was used to compare the continuous variables of autonomic neuropathy for treatment and placebo group. Multivariate correlation analysis was used to find the correlation between the neuropathy variables, biochemical parameters and diseases activity. A p value \0.05 were considered statistically significant. Statistical analysis was done using the Prism Graph Pad program for Windows 7.0.
Results A total of 50 type 2 diabetes patients eligible for the study gave informed consent to participate in the research study. The treatment group had 25 patients with mean age 58.50 ± 2.0 (9 females and 16 males) compared with 25 in the placebo group with mean age 60.46 ± 0.95 (7 females and 18 males). The demographic and laboratory features of treatment group and placebo group are summarized in Table 1. We found that there was no significant difference in the demographic and laboratory parameters of minocycline group and placebo group. The posttreatment changes in the neurological, CAN variables and sudomotor functions are shown in Table 2. Mean posttreatment score of VPT significantly lower in the minocycline group compared with the placebo group (22.29 ± 1.6 vs. 36.67. 1.6 ± 1.9, p = 0.01 vs. p = 0.80) (Table 2).
Table 1 The demographic and laboratory features of treatment and placebo group Minocycline (n = 25)
Placebo (n = 25)
p value
Age (years)
58.50 ± 2.0
60.46 ± 0.95
0.38
Sex (F/M)
9/16
7/18
–
Disease duration (years) BMI (kg/m2)
13.50 ± 1.48 26.29 ± 0.69
12.35 ± 0.84 25.17 ± 0.92
0.50 0.14 0.63
HbA1c (%)
7.15 ± 0.13
7.25 ± 0.15
Fasting sugar (mg/dL)
151.9 ± 13.31
159.6 ± 10.87
0.60
Systolic blood pressure (mm Hg)
131.8 ± 1.90
134.3 ± 1.68
0.34
Diastolic blood pressure (mm Hg)
82.08 ± 1.36
83.67 ± 1.36
0.41
Hb (g/Dl)
12.29 ± 0.29
12.36 ± 0.31
0.86
Uric acid (mg/dL) Serum creatinine (mg %)
5.24 ± 0.18 1.05 ± 0.04
5.02 ± 0.12 0.92 ± 0.02
0.33 0.02
Vitamin B12 (pg/mL)
635.8 ± 61.6
591.3 ± 53.4
0.58
Urine microalbuminuria
17.48 ± 0.99
15.87 ± 0.73
0.20
Data are means ± SEM. p \ 0.05 considered significantly F female, M male, BMI Body Mass Index
34.4 % of study patients treated with 6 weeks of minocycline improved while there was worsening of peripheral neuropathy in 0.06 % of patients in the placebo arm. LANSS and PDI were significantly (p B 0.01) improved in the minocycline group as compared to the placebo group (Table 2). However, BDI and VAS were significantly (p = 0.01) improved in both minocycline and placebo groups (Table 2). But the percentage improvement was higher in the minocycline group than in the placebo when improvement was measured on the BDI (53.98 vs. 23.94 %), and VAS (56.12 vs. 17.32 %) (Table 2). Heart rate (HR) response to standing significantly (p = 0.01) improved after treatment with minocycline (Table 2). After completion of the oral treatment period, there was a borderline significance toward a reduction in HR response to deep breath only in the minocycline-treated group as compared to the placebo-treated patients (Table 2). Other variables of CAN were not significantly improved in both minocycline- and placebo-treated group. Sudomotor function did not improve significantly in either minocycline-(p = 0.13) or placebo (p = 0.22)-treated diabetic patients (Table 2). After treatment with minocycline the quality of life assessed by HAQ was significantly improved in treatment group (62.74 %, p = 0.01) as compared to the placebo (25.45 %, p = 0.12) group (Table 2). There were a few minor number side effects in the minocycline group which included dizziness, nausea and vomiting. One patient complained of rashes following sun exposure.
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Neurol Sci Table 2 Effect of minocycline and placebo after 6 weeks of treatment on neurological variables, CAN, Sudoscan and HAQ in type 2 diabetic patients Variables
Minocycline 0 week
Group 6 week
p value
Placebo 0 week
Group 6 week
p value
34.00 ± 2.23
22.29 ± 1.65
\0.001
36.44 ± 1.94
36.67 ± 1.69
0.80
12.9 ± 1.12 14.8 ± 1.2
7.34 ± 0.12 8.3 ± 0.60
0.01 0.001
12.1 ± 0.02 14.13 ± 1.7
10.2 ± 1.23 11.92 ± 0.35
0.09 0.07
Neurological VPT LAANS PDI VAS
6.7 ± 0.11
2.9 ± 0.44
0.01
5.6 ± 0.22
4.63 ± 0.08
0.01
BDI
23.19 ± 0.35
10.67 ± 0.31
0.01
20.04 ± 0.54
14.96 ± 0.63
0.01
CAN variables HRV
1.26 ± 0.02
1.29 ± 0.04
0.16
1.22 ± 0.02
1.20 ± 0.03
0.14
HRD
9.54 ± 1.24
11.08 ± 1.04
0.06
10.08 ± 0.99
10.42 ± 0.90
0.45
HRS
1.00 ± 0.01
1.04 ± 0.06
0.01
0.97 ± 0.02
0.97 ± 0.02
0.08
BPS
7.75 ± 1.02
7.25 ± 0.89
0.37
8.58 ± 0.98
8.58 ± 0.98
0.90
BPH
14.25 ± 1.05
14.96 ± 5.15
0.45
13.13 ± 1.0
13.13 ± 1.06
0.78
Sudoscan
54.77 ± 3.96
59.34 ± 4.06
0.13
59.40 ± 2.7
59. 40 ± 2.76
0.22
1.9 ± 0.12
0.01
5.5 ± 1.3
HAQ
5.1 ± 1.2
4.1 ± 1.2
0.12
Data are means ± SEM. p \ 0.05 considered significantly VPT vibration perception threshold, LAANS Leeds Assessment of Neuropathic Symptoms and Signs, PDI Pain Disability Index, VAS Visual Analog Scale, BDI beck depression inventor, HRV HR response to valsalva, HRD HR response to deep breath, HRS HR response to standing, BPS BP response to standing, BPH BP response to handgrip, HAQ health assessment questionnaire
Changes in glycemic control of the patients and placebo group were followed for 6 weeks of treatment. In the control group, HbA1c (mean ± SEM) at baseline and after treatment was 7.25 ± 0.15 and 7.07 ± 0.16 % (p = 0.04), respectively. Corresponding values in the minocycline group was 7.15 ± 0.13 and 6.56 ± 0.25 % (p = 0.02), respectively. There were no significant differences between the two groups at baseline (Table 1). There was statistically significant correlation between VPT and HbA1c before (p = 0.02) and after (p = 0.01) minocycline therapy. VPT significantly correlated with BDI (p = 0.02), VAS (p = 0.03) heart rate response to deep breath (p = 0.004) and standing (p = 0.007) and Sudoscan (p = 0.001) after minocycline treatment. There was no statistically significant correlation among VPT and HbA1c, BDI, PDI, VAS, LANSS and autonomic neuropathy variables in placebo group.
Discussion Diabetic peripheral neuropathy and autonomic neuropathy are the most common neuropathies occurring in diabetes patients [1]. The early diagnosis and appropriate treatment of diabetic neuropathy is important because of patients being at higher risk of insensate injury to their feet due to DPN and also higher risk of morbidity and increased mortality, particularly if CAN is present [2]. As yet, there is no effective treatment to prevent the development and
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progression of human diabetic neuropathy. Therapeutic interventions that have been assessed include optimal glycemic control, aldose reductase inhibitors, angiotensin converting enzyme inhibitors, antioxidants (gamma linoleic acid, alpha lipoic acid, vitamin E) amino guanidine, advanced glycation end product inhibitor, C-peptide and Protein kinase C b inhibitor (which increase nerve blood flow) [2]. Some of these interventions have been withdrawn due to side effects and others are ineffective. Currently, duloxetine and pregabalin (both FDA approved) are the only medication that have been used for the treatment of peripheral neuropathic pain in diabetes [20]. The efficacies of minocycline on peripheral and autonomic neuropathy have not been previously investigated in type 2 diabetes while laboratory evidence supports its potential role in treatment of diabetic complications. An experimental animal study has reported a significant improvement in diabetic neuropathy after 3 weeks of treatment with minocycline in streptozotocin-induced diabetic neuropathy rats [7]. The results of the present study demonstrate that minocycline significantly improved VPT and painful diabetic neuropathy, i.e. LANSS and albeit small, improvement in HR response to standing and borderline significance toward HR response to deep breath. LANSS is an extremely good instrument to assess whether pain experienced is predominantly due to nerve damage or not. It is also the only published tool with validity for discriminating between neuropathic and nociceptive pain, regardless of the disease-based diagnostic methods [13].
Neurol Sci
The improvement in peripheral and autonomic neuropathy shown in this study over a very short period is more impressive than the improvement shown in trials of angiotensin converting enzyme inhibitor, aldose reductase inhibitors, alpha lipoic acid (ALADIN AND DEKAN study) and vitamin E [21–26], since minocycline has demonstrated a potential to improve both peripheral and autonomic neuropathies. The exact mechanisms by which minocycline exerted its effect on peripheral neuropathy are not known, but it could be related to its property to inhibit the enhanced production of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and reduction in glycemic control. The over production of these MMPs is responsible for the degradation or remodeling of the extracellular matrix (ECM) [26] and in addition, MMP-9 induces neuropathic pain through interleukin (IL)-1b cleavage and microglia activation, following nerve injury [27, 28]. This leads to damage of the vascular bed supplying nutritive blood to the sensory nerve fibers and responsible for peripheral nerve dysfunction in diabetes [28]. In the present study, we also observed that minocycline improved autonomic function as compared to the placebo group. There is recent evidence of activation of inflammatory cytokines IL-1 and IL-6 due to systemic inflammation [29]. Activation of these inflammatory cytokine significantly correlates with abnormalities in sympathetic vagal balance [29]. Minocycline possibly exerts its antiinflammatory effects through inhibition of inflammatory cytokines like IL-6, IL-1b, TNF-a and IL-8 [30–32] and reduction in mitogen-activated protein kinase (MAPK) pathways in microglial activation and proliferation [33]. Microglia are the primary immune effector cells of the central nervous system and play an important role in the initiation of inflammatory responses through inflammatory cytokines and chemokines [34, 35]. In this study, glycemic status improved in both the treatment and placebo groups, but improvement in minocycline group is much more than the placebo group. This is possibly due to inhibition of MMP-9 by minocycline [27]. In a recent study, HbA1c has been shown to be significantly associated with MMP-9 concentration in type 2 diabetes [36]. Hence, minocycline has a dual benefit in diabetic neuropathy. In this study, minocycline significantly improved LAANS, PDI score and VAS for pain. The improvement in LAANS, PDI score and VAS score suggests that minocycline could have the ability to improve neuropathic pain and improvement of glycemic status through inhibition of MMP-9 [27, 28]. Neuropathic pain is highly problematic for diabetic patients because it is often worse at night, depriving patients of sleep and thereby causing fatigue. In addition, it also has a strong negative effect on quality of
life (QOL) [20]. Interestingly, in the present study QOL assessed by HAQ significantly improved in minocycline group but not in placebo group. Patient VAS for pain also significantly improved in placebo group. The improvement in diabetic neuropathic pain in placebo group appears to be related to improvement in glycemic status since painful diabetic neuropathy is inversely related to the degree of glycemic control [37]. The percentage improvement in VAS is greater in minocycline group (56.12 %) as compared to placebo group (17.32 %) and also there was improvement in VPT, LAANS, PDI, QOL and HR response to standing in the study drug group only and not in the placebo group. It would thus appear that improvement in glycemic control alone cannot account for these improvements. In our study, we have found positive correlation between VPT and HbA1c before and after treatment with minocycline. Previous research has shown a positive association between impaired VPT and high HbA1c in diabetes [38] and our study results are also consistent with these results suggesting that patients with hyperglycemia may have higher risk of peripheral neuropathy. VPT also correlated with BDI, VAS, HR response to deep breath, HR response to standing and sudomotor function after minocycline treatment suggesting that improvement of DPN after therapy with minocycline is associated with improvement in depression, pain and autonomic dysfunction. In this study only two patients reported minor side effects, i.e. nausea, vomiting, dizziness and patient reported photosensitivity. These did not necessitate drug discontinuation. It should be noted that no particularly severe events were observed, thus confirming the safety of minocycline for long-term administration. Furthermore, we observed that the patient compliance was very high in this study because of close follow-up and regular contact with patients and also because of short follow-up of the study. Strength of this study is that it is the first to translate laboratory evidence to human experience; study subjects were well-matched with good overall selection with good treatment compliance and follow-up. Side effects were minor and did not necessitate discontinuation of study drug. Apart from primary benefits, the study demonstrated several ancillary benefits. The study limitation is that it included a small number of patients for short duration, and peripheral neuropathy evaluation in this study mainly relied on neuropsychological testing and used VPT LANSS, VAS, PDI and BDI as physical measures while autonomic dysfunction was characterized by an extensive battery of tests. In conclusion, this study demonstrates that treatment with minocycline is safe and well tolerated and significantly improves peripheral neuropathy and autonomic neuropathy in type 2 diabetes. It is important to point out
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that these effects might have a positive influence on cardiac risk associated with diabetic neuropathy. In addition minocycline appear to exert a beneficial effect on the glycemic status. The results of this pilot study would surely further research in this direction. Acknowledgments We wish to thank to the people who agreed to participate as subjects in the research reported here. We also gratefully acknowledge the availability of Sudoscan in our center by Impeto Medical, Paris, France. We are very thankful to University Grant Commission, New Delhi, India for providing the research fellowship [No. F.10-15/2007 (SA-I)]. Conflict of interest
None.
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