Clinical Trial of Nitrosense® Patch for the Treatment of Patients with Painful Diabetic Neuropathy

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Journal of the association of physicians of india • may 2014 • VOL. 62

Original Article

A Clinical Trial of Nitrosense® Patch for the Treatment of Patients with Painful Diabetic Neuropathy RP Agrawal1, S Jain2, S Goyal3, S Singhal4, L Lindgren5, E Sthengel6 Abstract Aims: Impaired nitric oxide synthesis has been implicated as one of the underlying causes of diabetic painful neuropathy (DPN). Hence, effects of a cutaneous, nitric oxide releasing patch (NitroSense® Derma Protect) were evaluated in subjects with DPN. Methods: Fifty diabetics were randomised to active/placebo arms after a 2 wk wash-out period. Patients received 24 mg patches (each patch releases around 9 nmol/cm 2/min of nitric oxide) for 3 hrs, every other day during a 3 wks period, or indistinguishable placebo patches. The extent of pain was recorded at start, at each visit and following completion of the study. Changes in pain from baseline were measured using the 11 point lickert scale (pls), visual analogue scale (vas), short form mcgill pain questionnaire (sf-mpq), present pain intensity (PPI) scale. Results: Subjects treated with patch experienced a statistically significant reduction in pain from baseline when compared to placebo (PLS scale; p = 0.05). Defining responders as subjects with a > 50% reduction in PLS score from baseline, the number needed to treat (NNT) was calculated as 3.0. A significant posttreatment decrease (p = 0.009) in vibration perception threshold (VPT) for left foot after active treatment was observed. Conclusions: Present results highlight utility of NitroSense® Derma Protect as controllable nitric oxide source for patients with DPN.

Introduction

D

iabetic painful neuropathy (DPN) is a major complication of both type 1 and type 2 diabetes mellitus. During the course of the condition, 20 to 90% of patients develop diabetic neuropathy 1 and 7.5% of diabetes patients present with neuropathy when diagnosed; this figure rises to 45% after 25 years. 2 In type 1 diabetes mellitus pain typically becomes symptomatic after years of chronic hyperglycaemia, while pain in patients with type 2 diabetes mellitus manifests within a few years of diagnosis. Professor, 2Research Scientist, Research Fellow, 4Statistician, Diabetes Care and Research Centre, S.P. Medical College, Bikaner, Rajasthan, India; 5 Director Research and Dev., 6 Laboratory Manager, NOLabs AB, Rönnowsgatan 8, S-252 25 Helsingborg, Sweden Received; 29.08.2012; Revised: 12.02.2013; Accepted: 02.07.2013 1

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DPN has a variable presentation with some patients experiencing painful neuropathy while others have asymptomatic, progressive loss of peripheral nerve function. 3 Pain in these patients manifests in various forms including burning, sharpness, tingling or deep lancinating pain and may develop into severe, unremitting pain with exacerbations at night. In more severe cases, patients also suffer from sleep deprivation and depression. 4 Several studies have implicated impaired nitric oxide synthesis, i.e. disturbance of endogenous nitric oxide production, as a contributing factor to the pathogenesis of DPN. In rats, impaired neuronal nitric oxide generation induced hyperalgesia, i.e. extreme sensitivity to pain. 5,6 There is also evidence that impaired blood flow plays a role and clinical data from type 2 diabetes patients with DPN has shown that decreased nitric oxide production influences endoneural blood flow. 7 The importance of nitric

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oxide in neuropathic pain is further substantiated by recent studies using various nitrate formulations, including, isosorbide dinitrate (ISDN) and glyceryl dinitrate (GTN). 8,9 NitroSense ® derma protect is a nitric oxide eluting patch which was registered as a Class I medical device for use as protective coverage to prevent the occurrence of ulcers on thinned skin in patients with diabetes or other diseases with skin involvement. Accordingly, NitroSense ® derma protect is applied on intact skin to directly cover and protect skin under the device. Pure nitric oxide is slowly released from the device, over several hours. In addition to its physical protective effects on skin, NitroSense ® derma protect is also protected from microbial invasion by nitric oxide. 10-12 A further effect of applying NitroSense ® derma protect to skin is the by nitric oxide mediated local vasodilatation that results in increased blood flow. In the proximity of hypoxic nerve endings found in the DPN patients, the nitric oxide mediated increase in blood flow might result in relief of neuropathic pain. The extent to which nitric oxide released from NitroSense ® derma protect directly applied to a diabetic foot exerts any effects has not previously been investigated. The primary objective of this study was to evaluate the feasibility of repeated NitroSense ® derma protect patch usage to protect skin on feet or at other locations in patients with diabetic neuropathy, including an assessment of the product’s safety. In addition, and considering the role of nitric oxide in the pathophysiology of neuropathy, the potential effects of nitric oxide on neuropathic pain relief were also studied as a secondary objective.

Subjects The study design was based on prior experience gained from similar studies using other nitric oxide releasing substances. 13-17 Results from these studies suggested that patients need at least three consecutive weeks of nitric oxide treatment in order to experience initial signs of significant decrease in neuropathic pain. Selection of Study Population

The present study was a double-blind, randomised, placebo controlled study enrolling 50 type 2 diabetes mellitus patients diagnosed with DPN (Clinical Investigation No. EX-260808-01). The trial complied with the Declaration of Helsinki and was approved by the Ethics Committee. Informed consent was obtained from all study participants. Inclusion Criteria

a. Male or female b. Age ≥ 18

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c. Stable glycaemic control d. No other medication for neuropathy for at least two weeks before inclusion e. At l e a s t t h r e e m o n t h s o f n e u r o p a t h i c p a i n symptom duration f. Peripheral neuropathy involving only lower limbs Exclusion Criteria

a. Erratic glycaemic control b. Peripheral vascular disease with absent foot pulses c. Presence of foot ulceration d. Treatment with sub lingual glyceryl trinitrate e. Male patient on sildenafil therapy or any other vasoactive medicine f. Presence of other causes of neuropathy like alcohol, renal impairment, drugs and toxins, nutritional deficiency g. Presence of other causes of pain h. Presence of skin disease, such as atopic dermatitis, psoriasis i.

Symptoms/diagnosis of depression

j. Patients with cardiac ischaemic and postural hypotension. Prior and Concomitant Therapy

Patients underwent a two week wash-out period if they were taking any prior medication for the treatment of neuropathic pain. Subjects were allowed to continue current drug therapy to manage diabetes. Use of pain medication during the study period was avoided, however, in case where rescue medication was needed, NSAIDs or paracetamol-based drugs were to be used. At the start, baseline information from anamnesis (medical history), neurological (including pain) and physical examinations, as well as from appropriate laboratory and physiological tests * were collected for each patient. Peripheral vascular disease was excluded based on clinical history and on patient’s ankle brachial index (ABI). Patients with adverse events including excessive skin reactions or allergic responses were removed from the study and not included in the assessment, as were the non-compliant patients.

Material and Method Treatment Administered

Patient treatments were performed at the Diabetes Care and Research Centre, SP Medical College, Bikaner, India and application time of NitroSense ® derma protect was controlled by a nurse administering * Blood glucose, blood urea, serum creatinine, HbA1c, lipid profile, urine for microalbuminuria, fundus, ECG and chest X-ray.

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Journal of the association of physicians of india • may 2014 • VOL. 62

Total Patients n=50

Two weeks washout period

Neurological examination Biochemical Investigations Pain assessment scores (SFMPQ, VAS, PPI and Lickert Scale) Nerve conduction velocity studies

Randomly selected Group A (Study group) n=25 NitroSense Derma Protect dressing (every second day / week for 3 weeks) on each foot dorsun

Group B (Placebo Group) n=25 Placebo dressing (every second day / week for 3 weeks) on each foot dorsum

Withdrawn due to skin reaction (n=1)

Withdrawn due to non compliance (n=1)

Completed trial (n=24)

Completed trial (n=24)

After 3 weeks Neurological examination Biochemical Investigations Pain assessment scores (SFMPQ, VAS, PPI and Lickert Scale) Nerve conduction velocity studies

treatment groups, and generated the randomisation list. The latter was done using dedicated computer software. In order to assure blinding of the study, special care was given to use a placebo patch with identical appearance to the active NitroSense ® derma protect patch. Dose Selection

Each NitroSense ® derma protect patch contains 24 mg of a nitric oxide donor. In-vitro measurements of release rates of the 24 mg NitroSense ® derma protect patch showed a release of 0.14 μmole of nitric oxide per minute or 9 nmole/cm 2 of nitric oxide per minute. Efficacy and safety assessments

To assess the safety of NitroSense ® derma protect, skin changes were evaluated by physical examination at the start, at pre-determined time points during the study, and at the end of the study. The efficacy of topical nitric oxide administered by NitroSense ® Derma Protect applied to skin was assessed on (1) neuropathic pain and (2) on the pathophysiology of diabetic neuropathy. Pain relief was measured using validated pain score methods, including visual analogue scale (vas ), present pain intensity (ppi ), 11 point lickert scale (p l s ), and the short form McGill pain questionnaire (SF-MPQ). Neurological examinations and nerve conduction studies were performed on the lower limb in all subjects at the start of the study and at follow-up after three weeks of usage of NitroSense ® derma protect. Neurological examination consisted of testing and grading of deep tendon reflexes, checking for sensory neuropathy using a Semmes-Weinstein monofilament 5.07 (10 gm) and vibration testing using a biothesiometer.

Statistical Methods Statistical Analysis (NNT; paired and unpaired students t’ test)

Fig. 1 : Flow chart

and removing the patch. Prior to each application, NitroSense® derma protect patches were prepared by a trained nurse or another trained medical professional according to instructions provided in the protocol. Treatment was initiated by applying NitroSense ® derma protect to the dorsum of the each foot or another painful area on each limb of a subject. Patches were left in situ for three hours there after NitroSense ® derma protect was removed by a nurse. Treatment was repeated every second week-day (i.e. Mondays, Wednesdays and Fridays), during three consecutive weeks (Figure 1). Methods of assigning patients to treatment group

An independent statistician provided the forcing block size, defined the patient distribution between

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Paired and un-paired students t-test were used for the statistical analysis of pain score (PLS, VAS, SFMPQ and PPI) and vibration perception threshold (VPT) data obtained from 48 subjects who completed the study. Statistical analyses were performed on different data sets comparing pain scores or VPT values at baseline (before treatment) to pain scores or VPT values after ten treatments (after three weeks).

Results A total of 50 subjects were enrolled in the study and 48 (96%) completed the three weeks of the study. After un-blinding there were 24 patients in each of active and placebo groups. One of the two subjects who did not complete the study was withdrawn due to a skin reaction; the other was excluded due to noncompliance. Except for the above mentioned subjects, no other complaints such as irritation or itching were reported. None of the subjects requested to change

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SFMPQ

PLS 0,5

1,0

0,0

0,0

-0,5

-1,0 -2,0

-1,0

-3,0

-1,5

-4,0

-2,0

-5,0 0

1

2

3

4

5

6

7

8

9

10

0

1

2

3

4

5

VAS

6

7

8

9

10

7

8

9

10

PPI

0,5

0,5

0,0 0,0

-0,5 -1,0

-0,5

-1,5

-1,0

-2,0 -2,5 0

1

2

3

4

5

6

7

8

9

10

-1,5 0

1

2

3

4

5

6

Fig. 2 : Comparisons of changes in pain scores over time (all subjects) each graph represents data from 48 patients assessed by one of the four different pain scales. Changes in pain score from baseline (y –axis) are plotted vs. time (number of visits, x-axis). Filled squares = placebo, Un-filled rhomboids = active Table 1 : Demographic and other baseline characteristics Parameter Number of patients Age, median (range) Weight (Kg), mean (range) Height (cm), mean (range) BMI, mean (range) Ankle brachial index HbA1c (%) Fasting blood sugar (mg/dl) Dyslipidaemia (mg/dl)

Active 24 52 (26 -75) 63.9 (44 – 88) 160,0 (144 – 176) 25.0 (17,2 – 32,9) R 1.21 (1.07-1.35), L 1.16 (0.62-1.37) 9.1 (5.6-13.4) 234.5 (93.2-442.6) 211.5 (148.5-344.2)

Placebo 24 55 (39 – 75) 66.1 (45 – 79) 161,3 (146 – 175) 25.9 (17,1 – 35,7) R 1.21 (0.96-1.38), L 1.18 (0.92-1.33) 9.67 (5.6-13.0) 239.36 (106.0-380.7) 197.93 (146.5-289.5)

the place of application, however, skin discolouration was observed in some cases. Despite no complaint by subjects, the place for application were changed and discolouration disappeared in 4-5 days.

There was a significant post-treatment decrease (from 27.50 ± 2.61 to 22.21 ± 2.21; p = 0.009) in VPT for the left foot following treatment with NitroSense® derma protect.

All subjects completing three weeks of NitroSense ® derma protect treatment experienced a reduction in pain (Table 2). Moreover, pain reduction was statistically significant when measured using the PLS scale, comparing active and placebo responses (p = 0.05, Table 2, Figure 2). Pain reduction as assessed by the VAS, SF-MPQ or PPI scales was not statistically significant. A subset analysis that excluded patients with mild pain (PLS < 4) showed a larger difference between active and placebo but the difference was still only significant for the PLS score, most likely due to a too low number of evaluated subjects (n = 17 active and 19 placebo, Table 3).

Analysis of the nerve conduction velocity did not show any significant difference among all groups analysed.

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The number needed to treat (NNT) was 3.0 when calculated using the change in PLS scores from baseline and defining responders as > 50% pain relief (Table 4). Only one patient receiving NitroSense ® derma protect experienced skin reaction which subsided spontaneously without any intervention.

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Table 2 : Comparison of pre- and post treatment pain score in the active and placebo arm Pre-treatment

Evaluation tool ACTIVE (n=24) PLS VAS SFMPQ PPI PLACEBO (n=24) PLS VAS SFMPQ PPI

PLS VAS SFMPQ PPI

Improvement from baseline

±se

Mean

±se

Diff

t

p*

5.08 5.38 31.04 2.79

0.41 0.42 3.26 0.21

3.42 3.63 27.29 1.71

0.33 0.28 3.03 0.19

-1.67 -1.75 -3.75 -1.08

7.78 5.04 3.10 6.84

£0.001 £0.001 0.0051 £0.001

0.35 3.88 0.27 0.39 3.58 0.32 3.26 22.67 2.38 0.14 1.96 0.14 Comparison of Active and Placebo response Active (n=24) Placebo (n=24) Mean Change Mean Change ±se ±se from baseline from baseline -1.67 0.21 -1.13 0.16 -1.75 0.35 -1.42 0.22

-1.13 -1.42 -2.21 -0.87

6.91 6.55 2.20 7.00

£0.001 £0.001 0.0384 £0.001

5.00 5.00 24.88 2.83

Evaluation tool

**

Post-treatment

Mean

-3.75 -1.08

1.21 0.16

-2.21 -0.88

1.01 0.13

Active placebo comparison Diff

t

p**

0.54 0.33

2.01 0.82

0.05 0.42

1.54 0.20

0.98 1.03

0.33 0.31

Unpaired, two-tailed t-test; *Paired, two-tailed t-test

Discussion Diabetic neuropathy is a symmetrical peripheral polyneuropathy that results from nerve damage after prolonged periods of suboptimal glycaemic control. 18 Diabetic neuropathy has been defined as presence of symptom and signs of peripheral nerve dysfunction in diabetes after exclusion of other causes, including hereditary, traumatic, compressive, metabolic, toxic, nutritional, infectious, immune mediated and neoplastic causes, as well as, causes secondary to other systemic illness. 19 In the absence of a curative therapy, treatment efforts are directed towards providing symptomatic pain control using pharmacological tools. Pharmacological therapy includes serotonin–norepinephrine reuptake inhibitors (duloxetine and velafaxine), tricyclic antidepressants (amitryptiline), narcotic analgesic (oxycodone CR), anticonvulsants (pregabalin, Gabapentin, carbamazepine and lamotrigine) and topical agents (capsaicin and lidocaine) but none of them is entirely satisfactory. 20,21 This present study is comparable to another study conducted using amitryptiline in which NNT was found to be 4.1. 22 Our previous double-blind, cross-over trial has demonstrated the utility of a GTN spray, for the treatment of pain due to DPN. In this study, 43 subjects with pain due to DPN were divided in to two cohorts. Cohort A received GTN for four weeks and cohort B received placebo. After two weeks, subjects in each cohort were crossed over and treatment continued for another four weeks. Overall, subjects in both cohorts experienced significant (p < 0.001)

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improvements in their pain scores while receiving GTN when compared to subjects receiving placebo. In addition, after crossing over into the GTN arm from placebo, subjects observed significant improvements (p < 0.001) in all pain scores. The NNT (using VAS scores) in this study was calculated as 4. The drug was well tolerated by all the patients except palpitation and headache for some days in five patients. 16 More recently we completed a study regarding clinical utility of topically applied nitrates for the treatment of DPN. For example, we have evaluated the safety and efficacy of sodium valproate and GTN alone, as well, as in combination in patients with DPN. In this study, 80 subjects were divided in to four cohorts, each receiving either GTN, sodium valproate, both or placebo. After three months, subjects in all three cohorts experienced significant (p < 0.001 to p < 0.05) improvements in pain scores along with improvements in certain electrophysiological parameters. The NNT (using VAS scores) for sodium valproate, GTN and their combination in this study were calculated as 7, 5 and 4, respectively. These results suggest combining drugs with different mechanisms of action may achieve synergistic analgesic effects in DPN. 17 Others have also observed antinociceptive effects of topically applied nitric oxide donors in patient with DPN. For example, glyceryl trinitrate (GTN), a nitric oxide donor with local vasodilatory effects, is bio-transformed to release nitric oxide that can activate guanylyl cyclase and increases the synthesis of cyclic-GMP in smooth muscle and other tissues. The pharmacological and biochemical effects of glyceryl

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Table 3 : Comparison between pain score improvement in active and placebo treatment groups. Only patients with severemoderate pain included in the comparison Evaluation tool PLS VAS SFMPQ PPI

Active (n=17) Mean change ±se from baseline -1.94 0.23 -2.35 0.33 -4.53 1.27 -1.24 0.17

Placebo (n=19) Mean change ±se from baseline -1.26 0.16 -1.47 0.23 -2.68 2.55 -0.89 0.13

Active placebo comparison Diff

t

p*

0.68 0.88 1.85 0.35

2.07 1.91 1.00 1.37

0.05 0.065 0.33 0.18

* un-paired, two-tailed t-test

Table 4 : % responders and NNT calculated for the active and placebo groups using the change in PLS score from baseline Whole group (n=48) Responders Active Placebo 62 % 29 % 29 % 0%

Criteria for positive outcome ≥ 30% reduction from baseline ≥ 50% reduction from baseline

trinitrate spray appear to be identical to those of endothelial-derived relaxing factor. 23 In summary, NitroSense derma protect is a highly promising device for the treatment of neuropathic pain in patients with DPN and additional studies should be conducted. The present study demonstrates the therapeutic utility of locally delivered nitric oxide. Furthermore, and in contrast to other topical nitric oxide formulations, NitroSense ® derma protect is safe, well tolerated and does not have any systemic side effects. An extended treatment with NitroSense ® derma protect to eight or more weeks, selecting patients with moderate to severe pain and/or better glycaemic control at baseline may result in even more pronounced pain relief. ®

Acknowledgement We are thankful to NO Labs AB, Sweden for supplying Nitrosense derma patch.

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