The adenosine deaminase inhibitor erythro-9-[2-hydroxyl-3-nonyl]-adenine decreases intestinal permeability and protects against experimental sepsis: a prospective, randomised laboratory investigation

Abstract

Introduction: The treatment of septic conditions in critically ill patients is still one of medicine's major challenges. Cyclic nucleotides, adenosine and its receptors play a pivotal role in the regulation of inflammatory responses and in limiting inflammatory tissue destruction. The aim of this study was to verify the hypothesis that adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibition by erythro-9-[2-hydroxyl-3-nonyl]-adenine could be beneficial in experimental endotoxicosis/sepsis.

Method: We used two established animal models for endotoxicosis and sepsis. Twenty-four male Wistar rats that had been given intravenous endotoxin (Escherichia coli lipopolysaccharide) were treated with either erythro-9-[2-hydroxyl-3-nonyl]-adenine infusion or 0.9% saline during a study length of 120 minutes. Sepsis in 84 female C57BL/6 mice was induced by caecal ligation and puncture. Animals were treated with repeated erythro-9-[2-hydroxyl-3-nonyl]-adenine injections after 0, 12 and 24 hours or 4, 12 and 24 hours for delayed treatment.

Results: In endotoxaemic rats, intestinal production of hypoxanthine increased from 9.8 +/- 90.2 micromol/l at baseline to 411.4 +/- 124.6 micromol/l and uric acid formation increased from 1.5 +/- 2.3 mmol/l to 13.1 +/- 2.7 mmol/l after 120 minutes. In endotoxaemic animals treated with erythro-9-[2-hydroxyl-3-nonyl]-adenine, we found no elevation of adenosine metabolites. The lactulose/L-rhamnose ratio (14.3 versus 4.2 in control animals; p = 2.5 x 10(-7)) reflects a highly permeable small intestine and through the application of erythro-9-[2-hydroxyl-3-nonyl]-adenine, intestinal permeability could be re-established. The lipopolysaccharide animals had decreased L-rhamnose/3-O-methyl-D-glucose urine excretion ratios. Erythro-9-[2-hydroxyl-3-nonyl]-adenine reduced this effect. The mucosa damage score of the septic animals was higher compared with control and therapy animals (p < 0.05). Septic shock induction by caecal ligation and puncture resulted in a 160-hour survival rate of about 25%. In contrast, direct adenosine deaminase-1 inhibition resulted in a survival rate of about 75% (p = 0.0018). A protective effect was still present when erythro-9-[2-hydroxyl-3-nonyl]-adenine treatment was delayed for four hours (55%, p = 0.029).

Conclusions: We present further evidence of the beneficial effects achieved by administering erythro-9-[2-hydroxyl-3-nonyl]-adenine, an adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibitor, in an endotoxicosis and sepsis animal model. This suggests a potential therapeutic option in the treatment of septic conditions.

Figure 1

Endotoxaemic challenge (experimental design). Rats were randomised to three groups of eight animals each. After preparation, a 30 minute stabilisation period was allowed. The animals of group B (lipopolysaccharide (LPS) + erythro-9-[2-hydroxyl-3-nonyl]-adenine (EHNA)) received 5 mg/kg/hour EHNA intravenously as a continuous infusion over one hour. Endotoxaemia was induced immediately after baseline measurements by continuous intravenous infusion of LPS for 60 minutes. Animals of the control group received no EHNA or LPS. The same amount of fluids was infused in all rats for the total duration of the experiment (120 minutes).

Figure 2

Adenosine deaminase-1 inhibition prevents lipopolysaccharide (LPS)-induced intestinal hypoxanthine and uric acid formation. (a) Intestinal release of hypoxanthine and (b) uric acid calculated as the differences (Δ) between portal venous and arterial concentrations of the purine metabolites after 120 minutes; in control animals, in animals receiving 1.5 mg/kg endotoxin over a 60 minute period (LPS group) and in animals receiving endotoxin plus an infusion of 5 mg/kg/hour erythro-9-[2-hydroxyl-3-nonyl]-adenine (EHNA) at the beginning of the endotoxin challenge (LPS + EHNA group). Data presented in one dimensional dot plots as well as mean and standard error of the mean (SEM). After 120 minutes the LPS group differed in the mean of intestinal hypoxanthine and uric acid production from control and EHNA-treated animals. (a) hypoxanthine production, analysis of variance (ANOVA) p = 0.02, post hoc Tukey-Kramer test p = 0.03; (b) uric acid production, ANOVA p = 0.01, post hoc Tukey-Kramer test p = 0.009.

Figure 3

Erythro-9-[2-hydroxyl-3-nonyl]-adenine (EHNA) administration re-establishes intestinal barrier as well as absorption capacity: Recovery of 3-O-methyl-D-glucose, lactulose and L-rhamnose in urine after direct duodenal administration was measured. (a) The lactulose/L-rhamnose ratio of the lipopolysaccharide (LPS) group was about three times higher than the control group, which indicates a highly permeable small intestine in septic rats. Through the application of EHNA the intestinal permeability could be re-established to a value comparable with control animals (analysis of variance (ANOVA) p = 3.5 × 10^-9, Tukey-Kramer test p = 2 × 10^-7). (b) LPS animals had decreased L-rhamnose/3-O-methyl-D-glucose urine excretion ratios (0.38 ± 0.05) compared with normal controls (0.58 ± 0.12, post hoc test p = 0.05), consistent with a decrease in the gastrointestinal functional absorptive capacity. ADA1 inhibition with a single dose of EHNA diminished this effect. Data presented in one dimensional dot plots as well as mean and standard error of the mean (SEM).

Figure 4

Adenosine deaminase inhibition protects against intestinal mucosal damage during endotoxaemia. Mucosal damage grading was assessed [31]. Data are mean ± standard error of the mean (SEM). # p < 0.05 versus control; $ p < 0.05 versus lipopolysaccharide (LPS) + erythro-9-[2-hydroxyl-3-nonyl]-adenine (EHNA).

Figure 5

Adenosine deaminase inhibition protects against intestinal mucosal damage during endotoxaemia. Representative microphotographs of haematoxylin & eosin (H & E) stained sections of the terminal ileum of experimental groups. (a,d) Control group with normal appearance of small intestinal mucosa with long villi that have occasional goblet cells, small and basal located nuclei of epithelial cells, and a normal lamina propria. (b, e) Lipopolysaccharide (LPS) group with disturb mucosal architecture showing plump villi with markedly increased villous stroma, a lifting of epithelial layer from the lamina propria (*subepithelial Gruenhagen's space), and a higher nucleus-plasma ratio of epithelial cells. (c, f) LPS + erythro-9-[2-hydroxyl-3-nonyl]-adenine (EHNA) group with a similar appearance of small intestinal mucosa as in the control group. (a-c) original magnification of ×16 and (d-F) ×64

Figure 6

Adenosine deaminase 1 inhibition protects against septic shock induced by caecal ligation and puncture (CLP). Kaplan-Meier survival curves; septic shock induction by CLP resulted in a 160-hour survival rate of about 25%. In contrast, direct adenosine deaminase-1 inhibition after septic shock induction via CLP resulted in a 160-hour survival rate of about 75% (p = 0.0018). A protective effect was still present when the erythro-9-[2-hydroxyl-3-nonyl]-adenine (EHNA) treatment was delayed for four hours after CLP (55% survival, p = 0.029).