Additional file 1: of The global intellectual property ecosystem for insulin and its public health implications: an observational study

Product Table: Patented products in North American market stratified by company and insulin analogue. (DOCX 15.2 KB

Additional file 2: of The global intellectual property ecosystem for insulin and its public health implications: an observational study

Analysis of patent searching using ‘insulin’ and/or ‘analogue/analog”. A. doc file with search results showing that using the word “analog” or “analogue” as an added search term does not increase the number of patent search result “hits”. (DOCX 131 KB

Additional file 2: of Trade in medicines and the public's health: a time series analysis of import disruptions during the 2015 India-Nepal border blockade

Residual calculation for penicillin and streptomycin exports. (XLSX 54 kb

Additional file 1: of Trade in medicines and the public's health: a time series analysis of import disruptions during the 2015 India-Nepal border blockade

Time-series analyses of trade in health commodities, 2011-2016. (PDF 801 kb

Reduced Retinal Microvascular Density, Improved Forepaw Reach, Comparative Microarray and Gene Set Enrichment Analysis with c-jun Targeting DNA Enzyme

<div><p>Retinal neovascularization is a critical component in the pathogenesis of common ocular disorders that cause blindness, and treatment options are limited. We evaluated the therapeutic effect of a DNA enzyme targeting c-jun mRNA in mice with pre-existing retinal neovascularization. A single injection of Dz13 in a lipid formulation containing N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine inhibited c-Jun expression and reduced retinal microvascular density. The DNAzyme inhibited retinal microvascular density as effectively as VEGF-A antibodies. Comparative microarray and gene expression analysis determined that Dz13 suppressed not only c-jun but a range of growth factors and matrix-degrading enzymes. Dz13 in this formulation inhibited microvascular endothelial cell proliferation, migration and tubule formation <em>in vitro</em>. Moreover, animals treated with Dz13 sensed the top of the cage in a modified forepaw reach model, unlike mice given a DNAzyme with scrambled RNA-binding arms that did not affect c-Jun expression. These findings demonstrate reduction of microvascular density and improvement in forepaw reach in mice administered catalytic DNA.</p> </div

Dz13 reduces pre-existing retinal microvascular density and penetrates the subretinal space.

<p>C57BL/6 mice were exposed to 75% oxygen for 4 d and returned to room air for 5 d. Dz13 or Dz13scr (20 µg in DOTAP/DOPE) was injected intravitreally (I-0). After 10 d (I-10), the animals were sacrificed, the eyes enucleated, cross-sectioned and stained with H&E (<b>A</b>) or, alternatively, with antibodies to CD34 (<b>B</b>)<b>.</b> Blood vessels on the vitreal side of the inner limiting membrane were quantified by counting erythrocyte-filled vessels in H&E-stained sections or CD34-stained vessels per section. “Blood vessel number” represents the mean number of blood vessels in 3 parallel cross-sections (5 µm each) per retina at 3 distances from the optic nerve (0, 50, 100 µm). Blood vessels are denoted by arrows. n = 6–8 eyes per group. Data is representative of 2 or more experiments. FITC-Dz13 (20 µg) in DOTAP/DOPE was injected intravitreally on I-0 and fluorescence intensity at I-2 was determined using NIH Image J software (<b>C</b>). *denotes P<0.05 relative to vehicle or Dz13scr.</p

Effect of Dz13 treatment in a modified forepaw reach model.

<p>C57BL/6 mice (I-0) were injected with Dz13 or Dz13scr (20 µg) in H₂O containing DOTAP/DOPE and on I-10, the animals were subjected to the forepaw reach reflex test. Mice forepaw-reaching reflex at the cage top was determined 5 times for each mouse. The observer was blinded to the groups. The circled mouse displays forepaw reach. n = 6–8 eyes per group. Data is representative of 2 or more experiments. * denotes P<0.05 relative to vehicle or Dz13scr.</p

Comparison of Dz13 with VEGF-A antibodies in the modified OIR model.

<p>(<b>A</b>) C57BL/6 mice (I-0) were injected with Dz13 (20 µg) in DOTAP/DOPE or its vehicle control (H₂O containing DOTAP/DOPE), VEGF-A antibodies (10 µg/µl) in PBS, pH 7.4 or IgG (10 µg/µl) in PBS, pH 7.4 and eyes removed at I-10. VEGF-A antibody concentration was as per Ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039160#pone.0039160-Aiello1" target="_blank">[23]</a>. Blood vessels on the vitreal side of the inner limiting membrane were quantified by counting erythrocyte-filled vessels in H&E-stained sections (<b>A</b>), CD34-stained sections (<b>B</b>) or c-Jun-stained section (<b>C</b>). “Blood vessel number” represents the mean number of blood vessels in 3 parallel cross-sections (5 µm each) per retina at 3 distances from the optic nerve (0, 50, 100 µm). n = 6–8 eyes per group. Blood vessels are denoted by arrows. *denotes P<0.05 relative to vehicle or IgG.</p

Dz13 inhibits c-Jun, FGF-2, VEGF-A, MMP-2 and MMP-9 expression in eyes of OIR mice.

<p>(<b>A</b>) Immunohistochemical analysis for c-Jun immunoreactivity in the retina of I-10 mice treated on I-0 with Dz13, Dz13scr or vehicle. Where indicated, IgG was used as the primary antibody as a negative control. Blood vessels are denoted by arrows. n = 6–8 eyes per group. (<b>B</b>) qPCR analysis on eyes of I-10 mice for c-jun, FGF-2, VEGF-A, VEGF-C, MMP-2, MMP-9, MMP-12 and MMP-13 mRNA. *denotes P<0.05 relative to vehicle and Dz13scr. Data is representative of 2 or more experiments.</p

Inhibition of endothelial cell proliferation, migration and tubule formation by Dz13 <i>in vitro</i>, and suppression of c-Jun and c-Jun-dependent gene expression.

<p>(<b>A</b>) Effect of Dz13 (0.4 µM) on serum-inducible HMEC proliferation after 2 d. Endothelial cell proliferation was quantified as the total number of cells per well (triplicate wells per group) in 96 well plates. (<b>B</b>) Toxicity and cell count in HMEC after 2 d. LDH activity and cell counts were quantified in triplicate cells of a 96 well plate per group. The % Control (FBS) for the “1-LDH” plot was calculated by subtracting LDH values (% LDH positive) in the Dz13-treated groups from 100% and expressing these as a percentage of LDH activity in the FBS control, and for the proliferation plot, expressing cell numbers the Dz13-treated groups as a percentage of numbers in the FBS control. (<b>C</b>) Effect of Dz13 (0.4 µM) on HMEC migration into the denuded zone (of 300 µm width) 2 d after <i>in vitro</i> scraping injury. Counts represent number of cells that have migrated into the denuded zone in 3 random fields using NIH Image J software. Calcein was added for 20 min before quantification. (<b>D</b>) Effect of Dz13 (0.2 µM) on tubule formation by HMEC on Matrigel. Tubules >3 µm in 96 well plates were quantified hourly in each of 4 random fields using NIH Image J software. Representative images taken at the 6 h time point are shown in the figure. (<b>E</b>) Western blotting using extracts of growth-quiescent HMEC exposed to 5% FBS for 1 h. SFM denotes serum-free medium. Blots were quantified by scanning densitometry. Data is representative of 2 or more experiments. *denotes P<0.05 relative to control.</p