Evaluation of the protein biomarkers and the analgesic response to systemic methylene blue in patients with refractory neuropathic pain: a double-blind, controlled study

Abstract

Aim: This study was carried out in patients with neuropathic pain in order to assess the analgesic effects and changes in protein biomarkers after the administration of methylene blue (MB), a diaminophenothiazine with antioxidant and anti-inflammatory properties, and with inhibitory effects on nitric oxide.

Materials and methods: Ten patients with chronic refractory neuropathic pain were randomized to receive either MB (10 mg/mL Methylthioninium chloride) 2 mg/kg (MB group) or MB 0.02 mg/kg (control group) infused over 60 minutes. Sensory function and pain (Numerical Rating Scale) were evaluated at baseline and at 60 minutes after the start of the infusion. The patients kept a pain diary during the next 24 hours and for the following 4 days. Plasma and urinary concentrations of 8-isoprostane-prostaglandin F2α (8-iso-PGF2α) and plasma protein biomarkers prior to and after the infusions were measured with radioimmunoassay and with proximity extension assay.

Results: A decrease of the Numerical Rating Scale at 60 minutes in comparison with baseline was observed in the MB (P=0.047) group. The decrease was significant between the MB and the control group on the day of and day after MB infusion (P=0.04 and P=0.008, respectively). There was no difference in systemic protein expressions between groups except for prolactin (PRL) (P=0.02). Three patients demonstrated diminished dynamic mechanical allodynia.

Conclusion: MB decreased the pain levels in patients with chronic therapy-resistant neuropathic pain on the first 2 days after administration. Known as an endocrine modulator on the anterior pituitary gland, MB infusion produced a decrease of PRL. The detailed role of PRL effects in chronic neuropathic pain remains undetermined.

Keywords: methylene blue; neuropathic pain; nitric oxide.

Figure 1

Numerical Rating Scale before and after the administration of MB (black) and in the control group (white). Note: The values are presented as the mean and 95% confidence intervals. Abbreviations: MB, methylene blue; h, hour.

Figure 2

Area of mechanical allodynia before MB (gray) and after MB (black) in patients 1 (postherpetic neuralgia), 3 (post-traumatic brachial injury), and 9 (central neuropathic pain after spinal cord ischemia). Abbreviation: MB, methylene blue.

Figure 3

Systolic blood pressure and oxygen saturation after MB administration (black circles) and control group administration (white circles). Notes: (A) Systolic blood pressure; (B) saturation. No differences were observed between the groups (P>0.5). Abbreviations: SABP, systolic arterial blood pressure; MB, methylene blue; min, minutes; SpO2, peripheral capillary oxygen saturation.

Figure 4

Principal component analysis comparing methylene blue (blue circles) with the control group (red circles). Notes: There is no clear clustering between the treatment groups. Ten proteins that contributed to the PCA were selected of 87 proteins (prolactin, TGF-α, TNF-RI, growth hormone, midkine, kallikrein-6, GM-CSF, cystatin B, CD69, and adrenomedullin). The significance level was P=0.3. Abbreviations: PCA, principal component analysis; TGF, transforming growth factor; TNF, tumor necrosis factor; GM-CSF, granulocyte macrophage colony-stimulating factor.

Figure 5

Principal component analysis somparing the before treatment proteins (green circles) with after treatment proteins in the methylene blue group (blue circles) and control group (red circles). Notes: There is some clustering observed before and after treatment. Fourteen proteins that contributed to the PCA were selected of 87 proteins (prolactin, TGF-α, TNF-RI, growth hormone, kallikrein-6, GM-CSF, CD30-L, thrombopoietin, prostasin, MMP-3, CXCL-11, EMMPRIN, HE4, and IFN-γ). The significance level was P=0.4. Abbreviations: PCA, principal component analysis. TGF, transforming growth factor; TNF, tumor necrosis factor; GM-CSF, granulocyte macrophage colony-stimulating factor; MMP, matrix metalloproteinase; CXCL, (C-X-C) motif ligand; IFN, interferon.