Metabolic interactions of benzodiazepines with oxycodone ex vivo and toxicity depending on usage patterns in an animal model.

BACKGROUND AND PURPOSE: Opioids and benzodiazepines are frequently combined in medical as well as in non-medical contexts. At high doses, such combinations often result in serious health complications attributed to pharmacodynamics interactions. Here, we investigate the contribution of the metabolic interactions between oxycodone, diazepam and diclazepam (a designer benzodiazepine) in abuse/overdose conditions through ex vivo, in vivo and in silico approaches. EXPERIMENTAL APPROACH: A preparation of pooled human liver microsomes was used to study oxycodone metabolism in the presence or absence of diazepam or diclazepam. In mice, diazepam or diclazepam was concomitantly administered with oxycodone to mimic acute intoxication. Diclazepam was introduced on Day 10 in mice continuously infused with oxycodone for 15 days to mimic chronic intoxication. In silico modelling was used to study the molecular interactions of the three drugs with CYP3A4 and 2D6. KEY RESULTS: In mice, in acute conditions, both diazepam and diclazepam inhibited the metabolism of oxycodone. In chronic conditions and at pharmacologically equivalent doses, diclazepam drastically enhanced the production of oxymorphone. In silico, the affinity of benzodiazepines was higher than oxycodone for CYP3A4, inhibiting oxycodone metabolism through CYP3A4. Oxycodone metabolism is likely to be diverted towards CYP2D6. CONCLUSION AND IMPLICATIONS: Acute doses of diazepam or diclazepam result in the accumulation of oxycodone, whereas chronic administration induces the accumulation of oxymorphone, the toxic metabolite. This suggests that overdoses of opioids in the presence of benzodiazepines are partly due to metabolic interactions, which in turn explain the patterns of toxicity dependent on usage. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Angiotensin IV improves subnormothermic machine perfusion preservation of rat liver graft.

This study aims to determine whether Angiotensin IV (Ang IV) addition to Celsior preservation solution could improve hepatic endothelium function and provide better liver protection during subnormothermic machine preservation (SMP). Two experimental models were used: In the first part of the study, rings isolated from rat hepatic artery were preserved in Celsior solution (6 h, 20 °C) with and without Ang IV (10-9 M), then, endothelium-dependent relaxation (EDR) and the concentration of acetylcholine inducing half-maximal relaxation of pre-contracted rings (EC50) were measured. Also, in order to investigate the implication of nitric oxide (NO) on EDR, the rings of hepatic artery were incubated with L-NG-nitroarginine metyl ester (L-NAME). In the second part of the study, rat livers were subjected to SMP with oxygenated Celsior solution (6 h, 20 °C), supplemented or not with Ang IV (10-9 M) and then perfused (2 h, 37 °C) with Krebs Henseleit solution. We found that Ang IV supplementation to Celsior solution decreased EC50 value and improved EDR of hepatic artery rings, 6h after sub-normothermic preservation. Interestingly, Ang IV amplified the vessel relaxation in a NO-dependent manner. Moreover, liver SMP with Ang IV reduced oxidative stress and cell injury and improved organ function. Ang IV activated pAkt, increased eNOS protein level and decreased apoptosis in the preserved liver grafts. In conclusion, we showed that the use of Ang IV in Celsior solution for sub-normothermic graft preservation insured a better NO-dependent relaxation and improved liver functional recovery.

Candesartan restores pressure-induced vasodilation and prevents skin pressure ulcer formation in diabetic mice.

BACKGROUND: Angiotensin II type 1 receptor (AT1R) blockers have beneficial effects on neurovascular complications in diabetes and in organ's protection against ischemic episodes. The present study examines whether the AT1R blocker candesartan (1) has a beneficial effect on diabetes-induced alteration of pressure-induced vasodilation (PIV, a cutaneous physiological neurovascular mechanism which could delay the occurrence of tissue ischemia), and (2) could be protective against skin pressure ulcer formation. METHODS: Male Swiss mice aged 5-6 weeks were randomly assigned to four experimental groups. In two groups, diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 200 mg.kg(-1)). After 6 weeks, control and STZ mice received either no treatment or candesartan (1 mg/kg-daily in drinking water) during 2 weeks. At the end of treatment (8 weeks of diabetes duration), C-fiber mediated nociception threshold, endothelium-dependent vasodilation and PIV were assessed. Pressure ulcers (PUs) were then induced by pinching the dorsal skin between two magnetic plates for three hours. Skin ulcer area development was assessed during three days, and histological examination of the depth of the skin lesion was performed at day three. RESULTS: After 8 weeks of diabetes, the skin neurovascular functions (C-fiber nociception, endothelium-dependent vasodilation and PIV) were markedly altered in STZ-treated mice, but were fully restored by treatment with candesartan. Whereas in diabetes mice exposure of the skin to pressure induced wide and deep necrotic lesions, treatment with candersartan restored their ability to resist to pressure-induced ulceration as efficiently as the control mice. CONCLUSION: Candesartan decreases the vulnerability to pressure-induced ulceration and restores skin neurovascular functions in mice with STZ-induced established diabetes.

Effects of Institut Georges Lopez-1 and Celsior preservation solutions on liver graft injury.

AIM: To compare Institut Georges Lopez (IGL-1) and Celsior preservation solutions for hepatic endothelium relaxation and liver cold ischemia reperfusion injury (IRI). METHODS: Two experimental models were used. In the first one, acetylcholine-induced endothelium-dependent relaxation (EDR) was measured in isolated ring preparations of rat hepatic arteries preserved or not in IGL-1 or Celsior solutions (24 h at 4 °C). To determine nitric oxide (NO) and cyclooxygenase EDR, hepatic arteries were incubated with L-NG-nitroarginine methyl ester (L-NAME), an inhibitor of endothelium nitric oxide synthase (eNOS), or with L-NAME plus indomethacin, an inhibitor of cyclooxygenase. In the second experiment, rat livers were cold-stored in IGL-1 or Celsior solutions for 24 h at 4 °C and then perfused "ex vivo" for 2 h at 37 °C. Liver injury was assessed by transaminase measurements, liver function by bile production and bromosulfophthalein clearance, oxidative stress by malondialdehyde levels and catalase activity and alterations in cell signaling pathways by pAkt, pAMPK, eNOS and MAPKs proteins level. RESULTS: After cold storage for 24 h with either Celsior or IGL-1, EDR was only slightly altered. In freshly isolated arteries, EDR was exclusively mediated by NO. However, cold-stored arteries showed NO- and COX-dependent relaxation. The decrease in NO-dependent relaxation after cold storage was significantly more marked with Celsior. The second study indicated that IGL-1 solution obtained better liver preservation and protection against IRI than Celsior. Liver injury was reduced, function was improved and there was less oxidative stress. IGL-1 solution activated Akt and AMPK, which was concomitant with increased eNOS expression and nitrite/nitrate levels. Furthermore, MAPKs kinases were regulated in livers preserved with IGL-1 solution since reductions in p-p38, p-ERK and p-JNK protein levels were observed. CONCLUSION: IGL-1 solution preserved NO-dependent relaxation better than Celsior storage solution and enhanced liver graft preservation.

Opposite effects of angiotensins receptors type 2 and type 4 on streptozotocin induced diabetes vascular alterations in mice.

BACKGROUND: We examined the effect of chronic administration of angiotensin IV (AngIV) on the vascular alterations induced by type 1 diabetes in mice. METHODS: Diabetes was induced in adult Swiss mice with a single injection of streptozotocin (STZ). Mice were treated subcutaneously with AngIV (1.4 mg/kg/day) either immediately following diabetes induction (preventive treatment), or treated with AngIV (0.01 to 1.4 mg/kg), alone or with the AT4 receptor antagonist Divalinal or the AT2 receptor antagonist PD123319, for two weeks after 4 weeks of diabetes duration (rescue treatment). Acetylcholine-induced, endothelium-dependent relaxation (EDR) was measured in isolated aortic rings preparations. Histomorphometric measurements of the media thickness were obtained, and nitric oxide (NO) and superoxide anion production were measured by electron paramagnetic resonance in aorta and mesenteric arteries. The effect of diabetes on mesenteric vascular alterations was also examined in genetically modified mice lacking the AT2 receptor. RESULTS: Induction of diabetes with STZ was associated with a progressive decrease of EDR and an increase of the aortic and mesenteric media thickness already significant after 4 weeks and peaking at week 6. Immediate treatment with AngIV fully prevented the diabetes-induced endothelial dysfunction. Rescue treatment with AngIV implemented after 4 weeks of diabetes dose-dependently restored a normal endothelial function at week 6. AngIV blunted the thickening of the aortic and mesenteric media, and reversed the diabetes-induced changes in NO and O2•- production by the vessels. The protective effect of AngIV on endothelial function was completely blunted by cotreatment with Divalinal, but not with PD123319. In contrast, both the pharmacological blockade and genetic deletion of the AT2 receptor reversed the diabetes-induced morphologic and endothelial alteration caused by diabetes. CONCLUSIONS: The results suggest an opposite contribution of AT2 and AT4 receptors to the vascular alterations caused by streptozotocin-induced diabetes in mice, since chronic stimulation of AT4 by AngIV and inhibition of AT2 similarly reverse diabetes-induced endothelial dysfunction and hypertrophic remodeling, and increase NO bioavailability.

Erythropoietin restores C-fiber function and prevents pressure ulcer formation in diabetic mice.

Pressure-induced vasodilatation (PIV), a cutaneous physiological neurovascular (C-fiber/endothelium) mechanism, is altered in diabetes and could possibly contribute to pressure ulcer development. We wanted to determine whether recombinant human erythropoietin (rhEPO), which has protective neurovascular effects, could prevent PIV alteration and pressure ulcer formation. We developed a skin pressure ulcer model in mice by applying two magnetic plates to the dorsal skin. This induced significant stage 2 ulcers (assessed visually and histologically) in streptozotocin-treated mice with 8 weeks of diabetes compared with very few in controls. Control and streptozotocin mice received either no treatment or systematic rhEPO (3,000 UI kg(-1) intraperitoneally, twice a week) during the last 2 weeks of diabetes. After 8 weeks of diabetes, we assessed ulcer development, PIV, endothelium-dependent vasodilation, C-fiber-mediated nociception threshold, and skin innervation density. Pretreatment with rhEPO fully prevented ulcer development in streptozotocin mice and also fully restored C-fiber nociception, skin innervation density, and significantly improved PIV, but had no effect on endothelium-dependent vasodilation. Our finding that rhEPO treatment protects the skin against pressure-induced ulcers in diabetic mice encourages evaluation of the therapeutic potential for non-hematopoietic analogs of EPO in preventing neuropathic diabetic ulcers.

Protective effect of candesartan in experimental ischemic stroke in the rat mediated by AT2 and AT4 receptors.

OBJECTIVES: The contribution of the AT2 and AT4 angiotensin receptors to the protective role of the AT1 receptor blocker candesartan in acute ischemic stroke was investigated. METHODS: Embolic stroke was induced by injection of calibrated microspheres (50 microm) in the right internal carotid in Sprague-Dawley rats. RESULTS: Inhibition of production of endogenous angiotensins by pretreatment for 24 h with lisinopril significantly increased mortality and infarct volume, whereas candesartan for 24 h reduced blood pressure to the same extent but had no deleterious effect. A more sustained pretreatment with candesartan for 5 days significantly decreased mortality, neurological deficit and infarct size. The AT2 receptor antagonist PD123319 and the AT4 receptor antagonist divalinal abolished the protective effect of 5 days' AT1 blockade. Combined blockade of AT2 and AT4 in candesartan pretreated rats resulted in an increased mortality, neurological deficit and infarct volume of similar magnitude to lisinopril pretreatment. Coadministration of lisinopril 24 h before surgery completely blunted the protective effect of candesartan pretreatment. Administration of exogenous angiotensin IV (1 nmol) reversed the deleterious effect of lisinopril pretreatment. CONCLUSION: Protection against acute cerebral ischemia induced by AT1 blockade for 5 days is blood pressure independent and mediated by both AT2 and AT4 angiotensin receptors.

Synergistic protective effects of erythropoietin and olmesartan on ischemic stroke survival and post-stroke memory dysfunctions in the gerbil.

OBJECTIVES: Treatment with erythropoietin and AT1 blockers is protective in experimental acute cerebral ischemia, with promising results in pilot clinical studies in human stroke. This paper examines the effects of using both agents as combination therapy in acute ischemic stroke. METHODS: We used the single carotid ligation stroke model in the gerbil. Six groups of 50 gerbils were treated either with placebo, erythropoietin (intraperitoneally, 5000 IU/kg, 2 and 48 h after stroke), olmesartan (10 mg/kg per day in drinking water started 36 h after stroke), ramipril (2.5 mg/kg per day in drinking water started 36 h after stroke), erythropoietin + olmesartan, or erythropoietin + ramipril. Long-term (1 month) Kaplan-Meyer survival curves were obtained, and survivors were submitted at day 30 to immediate (object recognition test) and spatial (Morris water maze) memory function tests. RESULTS: Erythropoietin alone and olmesartan alone, but not ramipril, significantly increased survival at day 30 compared with untreated controls (38, 30 and 6% versus 12%, respectively). Combined treatment with erythropoietin and olmesartan further increased the survival rate to 56%, whereas combined therapy with erythropoietin and ramipril decreased 30-day survival to 24% (P < 0.0001, erythropoietin + olmesartan versus erythropoietin + ramipril). Untreated stroke survivors had markedly altered performances in both the object recognition test (P = 0.0007) and the Morris water maze (P < 0.0001) tests at day 30 compared with normal gerbils. In erythropoietin-treated animals, ramipril therapy had no beneficial effect whereas olmesartan fully restored normal response to the memory tests. CONCLUSION: Post-infarct treatment with olmesartan combined with early erythropoietin therapy has a protective effect on survival, and markedly improves long-term memory dysfunction in this experimental model.

Vasoconstrictive effect of angiotensin IV in isolated rat basilar artery independent of AT1 and AT2 receptors.

The effect of angiotensin IV (AngIV) was studied in freshly isolated rat basilar arteries (BAs) perfused at a constant rate. AngIV had no effect on basal BA perfusion pressure, but induced a marked concentration-dependent contraction in vessels precontracted by a 50-mM KCl solution (EC50=44.5+/-16 nM). This contraction was unaffected by the angiotensin AT1 receptor antagonist candesartan or the angiotensin AT2 receptor blocker PD123319, but was markedly inhibited by two different specific AT4 receptor antagonists, Nle1-Leu3 yen(CH2-NH2)3-4-AngIV and divalinal-AngIV. Removal of the endothelium abolished the contractile response to AngIV, and pretreatment of endothelium-intact arteries with the endothelin ETA/ETB receptors inhibitor PD142893 blocked the AngIV-induced contraction to the same extent. In BA pretreated with endothelin-1 (ET-1; 0.01 microM), AngIV-induced a concentration-dependent contraction, shifted to the left, compared with that observed with KCl precontraction, unaffected by candesartan but completely abolished by Nle1-Leu3 yen(CH2-NH2)3-4-AngIV. The contractile effect was not affected by endothelium removal in the presence of exogenous ET-1, in contrast to KCl pretreated BA, suggesting that endothelium was mandatory to unmask the effect of AngIV as a source of endogenous ET-1 release. Taken together, these results indicate that low (nanomolar) concentrations of AngIV exert a constrictive effect mediated by its specific binding site AT4 in the rat BA, and that this vasoactive effect is indirect and involves endogenous endothelin(s).