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Was comparable to the hexasaccharide fraction (Figure 2, 3). Figure 4 shows that the anti-allergic activity of the tetrasaccharide fraction was dose-dependent. While 0.03 mg/kg (n = 5) dose was ineffective, the 0.06 mg/kg (n = 6) and 0.125 mg/kg (n = 5) doses inhibited the EAR AUC0-4h by 47 ?13 and 63 ?11 (P < .05); LAR AUC4-8h by 63 ?7 and 81 ?9 (P < .05). Antigen-induced AHR at these doses was inhibited by 95 ?9 and 100 ?10 (P < .05); respectively.Post-antigen administration of TetrasaccharidesInhaled tetrasaccharide (n = 5) caused a dose-dependent inhibition of LAR AUC4-8h and AHR when administered "after" the antigen challenge (Figure 5). While 0.03 mg/kg dose was ineffective, post-antigen administration of 0.06 mg/kg tetrasaccharide inhibited the LAR AUC4-8hAhmed et al. Respiratory Research 2012, 13:6 http://respiratory-research.com/content/13/1/Page 6 ofDUAL RESPONDERS500OLIGOSACCHARIDESRL ( CHANGE)400 300 200 100 0 500 400 Bsl 0 1 2 3 4 5 6 6.5 7 7.50.5 mg/kg400 300 200 100 0 500 400 300 200 100 0 Bsl 0OCTASACCHARIDE 0.5 mg/kg6 6.5 7 7.5SRL ( CHANGE)HEXASACCHARIDE 0.125 mg/kgTETRASACCHARIDE 0.125 mg/kg300 200 100 0 Bsl 0 1 2 3 4 5 6 6.5 7 7.5 8 HOURS POST ANTIGENBsl6 6.5 7 7.5HOURS POST ANTIGENFigure 2 Effect of inhaled heparin oligosaccharides on antigen-induced early and late airway responses (EAR and LAR) in "Dual Responder" sheep. Data are shown as change in specific lung resistance (SRL) ?SE without (solid circles) and 3-Amino-1H-indazole-4-carbonitrile after (open circles) pretreatment with oligosaccharide mixture (n = 6; 0.5 mg/kg), and various fractions including octasaccharide (n = 3; 0.5 mg/kg), hexasaccharide (n = 5; 0.125 mg/kg), and tetrasaccharide (n Boc-D-Lys-OH = 5; 0.125 mg/kg). Bsl = Baseline.OLIGOSACCHARIDEPD400 (BREATH UNITS)0.5 mg/kg+OCTASACCHARIDE 0.5 mg/kg+25 20 15 10 5 025*15 10 5*PD400 (BREATH UNITS)25 20 15 10 5HEXASACCHARIDE 0.125 mg/kg +30 25TETRASACCHARIDE + 0.125 mg/kg*15 10*BASELINE ANTIGENDRUGBASELINE ANTIGENDRUGFigure 3 Effect of inhaled heparin oligosaccharides on antigen-induced airway hyperresponsiveness (AHR) in "Dual Responder" sheep. Data are shown as PD400 (cumulative provocating dose of carbachol in breath units which increased SRL by 400 ) ?SE for the baseline, and 24 hours post-antigen, without and after pretreatment with oligosaccharide mixture (0.5 mg/kg) and various fractions including Octasaccharide (0.5 mg/kg), hexasaccharide (0.125 mg/kg) and tetrasaccharide (0.125 mg/kg). * Significantly different from baseline (P < .05). + Significantly different from antigen control (P < .05).Ahmed et al. Respiratory Research 2012, 13:6 http://respiratory-research.com/content/13/1/Page 7 of500 SRL ( PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17591728 CHANGE) 400 300 200 100TETRASACCHARIDE (PRE-ANTIGEN ) SRL ( CHANGE)0.03 mg/kg500 400 300 200 100TETRASACCHARIDE (POST ANTIGEN) P0.03 mg/kgBSL 0 500 SRL ( CHANGE)6 6.5 7 7.5BSL 06.7.SRL ( CHANGE)400 300 200 100 0 BSL 0 500 1 2 3 40.06 mg/kg400 300 200 100 0 BSL 0 500 1 2 3 40.06 mg/kg6 6.5 7 7.56.7.SRL ( CHANGE)SRL ( CHANGE)400 300 200 100 0 BSL 0 1 2 3 40.125 mg/kg400 300 200 100 0 BSL 0 1 2 3 40.125 mg/kg6 6.5 7 7.56.7.HOURS PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 POST ANTIGENHOURS POST ANTIGENFigure 4 Dose response effect of pretreatment with inhaled heparin tetrasaccharide on antigen-induced early and late airway responses (EAR and LAR) in "Dual Responder" sheep. Data are shown as antigen-induced change in specific lung resistance (SRL) ?SE without (solid circles) and after pretreatment (open circles) with inhaled tetrasaccharide (0.03 mg/kg, 1-dimethylethyl ester 4-Bromo-3 n = 5); 0.06 mg/kg,.