Amira M. Senbel

A Novel COX‐2 Inhibitor Pyrazole Derivative Proven Effective as an Anti‐Inflammatory and Analgesic Drug

The introduction of new COX-2 inhibitors with high efficacy and enhanced safety profile would be a great achievement in the development of anti-inflammatory drugs. This study was designed to screen and assess the anti-inflammatory and analgesic activities as well as some of the expected side effects of some pyrazole derivatives, newly synthesized as potential COX-2 inhibitors at the Faculty of Pharmacy, Alexandria University and compared to indomethacin and celecoxib. Twelve compounds were screened for their anti-inflammatory activity using carrageenan-induced paw oedema and cotton pellet granuloma tests. On the basis of their apparent anti-inflammatory activity, four compounds with different substitutions were selected for the evaluation of their analgesic activity using the formalin-induced hyperalgesia and hot-plate tests. Compound AD 532, ((4-(3-(4-Methylphenyl)-4-cyano-1H-pyrazol-1-yl)benzenesulfonamide)), showed very promising results. In the single-dose and subchronic toxicity studies, compound AD 532 showed no ulcerogenic effect and produced minimal effects on renal function. Furthermore, compound AD 532 was a less potent inhibitor of COX-2 in vitro than celecoxib, which may indicate lower potential cardiovascular toxicity. It is concluded that compound AD 532 appears to be a promising and safe option for the management of chronic inflammatory conditions. This study recommends more in-depth investigation into the therapeutic effects and toxicity profile of this compound including its cardiovascular toxicity.

Effect of mesenchymal stem cell penile transplantation on erectile signaling of aged rats

Stem cell‐based therapy targeted at the penile tissue has been lately considered in preclinical studies. This work aimed to assess the effect of intracavernous administration of mesenchymal stem cells (MSCs) in aged rats (n = 100). They were subjected to single intracavernous injection (ICI) of 1.0 million MSCs, followed up for 3, 4 weeks, 3 and 4 months (each group 25 rats) and compared with both adult and aged controls (n = 50). In dissected cavernous tissues, cGMP and histopathology were assessed in addition to intracavernous pressure (ICP) measurement in some anaesthetised rats. The results showed that cavernous tissue cGMP was significantly increased in MSCs transplanted rats in all investigated groups compared with the controls. The mean cavernous cGMP levels after 3 and 4 months of MSCs transplantation were significantly increased compared with those after 3 or 4 weeks. Cavernous tissue ICP measurement showed significant increase in MSCs transplanted groups compared with the controls, more in the long‐term follow up than in the shorter one. Histopathological examination detected markedly dilated sinusoidal vascular spaces in the long‐term follow‐up study. It is concluded that stem cell‐based therapy is feasible for age‐associated erectile dysfunction and could improve erectile signaling.

Effects of Losartan, HO‐1 Inducers or HO‐1 Inhibitors on Erectile Signaling in Diabetic Rats

INTRODUCTION Activation of the renin-angiotensin system which is common in diabetes mellitus might affect heme oxygenase (HO-1) gene expression. AIM Assessment of the effects of administration of angiotensin II (Ang II) receptor antagonist (losartan) with HO-1 inducer or inhibitor on erectile signaling in diabetic rats. MATERIALS AND METHODS Seventy male rats were divided equally into seven groups; healthy controls, streptozotocin-induced diabetic rats, rats on citrate buffer, diabetic rats on losartan, diabetic rats on HO-1 inducer (cobalt protoporphyrin [CoPP]), diabetic rats on losartan and CoPP, and diabetic rats on losartan and HO-1 inhibitor (stannus mesoporphyrin [SnMP]). MAIN OUTCOME MEASURE HO enzyme activity, HO-1 gene expression, cyclic guanosine monophosphate (cGMP) assay, intracavernosal pressure (ICP), and cavernous tissue sinusoids surface area. RESULTS HO-1 gene expression, HO enzymatic activity, and cGMP were significantly decreased in the cavernous tissue of diabetic rats. These parameters were significantly elevated with the use of CoPP that restored the normal control levels of HO enzyme activity. Administration of losartan exhibited a significant enhancing effect on these parameters compared with the diabetic group, but not restored to the control levels, whereas administration of CoPP combined with losartan led to the restoration of their normal levels. ICP demonstrated significant decline in diabetic rats. The use of CoPP and/or losartan led to its significant improvement compared with diabetic rats. Administration of either losartan and/or CoPP led to a significant increase in the cavernous sinusoids surface area of diabetic rats. Administration of losartan with SnMP significantly decreased the enhancing effect of losartan on the studied parameters. CONCLUSION The decline in erectile function in diabetes mellitus could be attributed to the downregulation of HO-1 gene expression. HO-1 induction added to Ang II receptor antagonist could improve erectile function.

Celecoxib modulates nitric oxide and reactive oxygen species in kidney ischemia/reperfusion injury and rat aorta model of hypoxia/reoxygenation.

OBJECTIVE This study investigated the interaction between COX-2, NO and ROS after ischemia/reperfusion events in the kidney and vascular beds. MATERIALS AND METHODS Kidney IRI model in male Sprague-Dawley rats was used and various biochemical and histopathological parameters were examined. The isolated rat aortic rings served as model for hypoxia/reoxygenation. RESULTS Celecoxib reduced serum creatinine and urea and kidney malonaldehyde levels, increased kidney superoxide dismutase activity and reduced glutathione level and histopathological scores at 24 and 48 h after reperfusion compared to IRI group. This was associated with a significant increase in NO level to 0.70 ± 0.03 nmol/mg protein compared to 0.37 ± 0.01 nmol/mg protein for IRI group. Unexpectedly, celecoxib reduced COX-2 expression in the kidney. Celecoxib reversed the effect of hypoxia-reoxygenation on ACh and SNP-induced relaxation in aortic rings but failed to potentiate the SNP relaxations in the control rings. Hypoxia-reoxygenation significantly impaired celecoxibs relaxation of aorta (12.69 ± 2.69% vs. 35.84 ± 0.84%) which was significantly inhibited in presence of L-NAME. CONCLUSIONS Celecoxib beneficially affects the outcome of renal IRI by lowering the expression of COX-2 and hence reducing oxidative stress and increasing the bioavailability of NO. Direct interaction between celecoxib and NO in associated vascular beds may also be a contributing mechanism.

Effects of a Water‐Soluble Curcumin Protein Conjugate vs. Pure Curcumin in a Diabetic Model of Erectile Dysfunction

INTRODUCTION Curcumin is involved in erectile signaling via elevation of cyclic guanosine monophosphate (cGMP). AIM Assessment of the effects of water-soluble curcumin in erectile dysfunction (ED). METHODS One hundred twenty male white albino rats were divided into: 1st and 2nd control groups with or without administration of Zinc protoporphyrin (ZnPP), 3rd and 4th diabetic groups with or without ZnPP, 5th diabetic group on single oral dose of pure curcumin, 6th diabetic group on pure curcumin administered daily for 12 weeks, 7th and 8th diabetic groups on single dose of water-soluble curcumin administered with or without ZnPP, 9th and 10th diabetic groups on water-soluble curcumin administered daily for 12 weeks with or without ZnPP. All curcumin dosage schedules were administered after induction of diabetes. MAIN OUTCOME MEASURES Quantitative gene expression of endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), heme oxygenase-1 (HO-1), nuclear transcription factor-erythroid2 (Nrf2), NF-Кβ, and p38. Cavernous tissue levels of HO and NOS enzyme activities, cGMP and intracavernosal pressure (ICP). RESULTS Twelve weeks after induction of diabetes, ED was confirmed by the significant decrease in ICP. There was a significant decrease in cGMP, NOS, HO enzymes, a significant decrease in eNOS, nNOS, HO-1 genes and a significant elevation of NF-Кβ, p38, iNOS genes. Administration of pure curcumin or its water-soluble conjugate led to a significant elevation in ICP, cGMP levels, a significant increase in HO-1 and NOS enzymes, a significant increase in eNOS, nNOS, HO-1, and Nrf2 genes, and a significant decrease in NF-Кβ, p38, and iNOS genes. Water-soluble curcumin showed significant superiority and more prolonged duration of action. Repeated doses regimens were superior to single dose regimen. Administration of ZnPP significantly reduced HO enzyme, cGMP, ICP/ mean arterial pressure (MAP), HO-1 genes in diabetic groups. CONCLUSION Water-soluble curcumin could enhance erectile function with more effectiveness and with more prolonged duration of action.

Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats

AIMS The present study aims to elucidate the role of oxidative stress in erectile dysfunction associated with aging and to investigate the effect of treatment with vitamin E in this respect. MAIN METHODS Rats were divided into four groups: young (3-month-old), aged rats (18-month-old), aged rats given 80 IU of vitamin E/rat/day for 21-days, aged rats given 5mg/kg of sildenafil/day for 21-days. Intracavernosal pressure/mean arterial pressure (ICP/MAP), nitric oxide production, TBARS, GSH levels and SOD activity in corpus cavernosum were measured. KEY FINDINGS Significant decrease in ICP/MAP was observed in aged rats at both low and high frequency of stimulation. Significant increase in ICP/MAP was observed in aged rats treated with vitamin E over the range of 0.8 to 5 Hz but young control values were not restored. Percentage potentiation of ICP/MAP than aged group at 0.8 Hz was 326±41.3% and 897±72.2% for vitamin E and sildenafil respectively. Decreased levels of NO(2)/NO(3) and SOD activity in the penile tissue observed with aging were elevated back to control by either vitamin E or sildenafil. Penile concentration of TBARS was 20.86±0.83 for aged rats vs. 11.39±0.79 nmol/g tissue for young controls. Both vitamin E and sildenafil reduced penile TBARS in aged rats. SIGNIFICANCE This study proves that antioxidant therapy with vitamin E ameliorates the age-associated erectile dysfunction. Sildenafil may exert some antioxidant properties which add to the advantages of its long-term use. The effect of combinations of low-dose sildenafil and vitamin E on age-associated erectile dysfunction merits to be studied.

Comparative effects of sildenafil, phentolamine, yohimbine and L-arginine on the rabbit corpus cavernosum.

Penile erection involves relaxation of smooth muscle of corpus cavernosum and associated arterioles. Sildenafil, a highly selective inhibitor of phosphodiesterase type 5, is effective in the treatment of erectile dysfunction. The aim of this study is to investigate the effect of sildenafil on smooth muscle of the rabbit corpus cavernosum (RCC) and to compare its effect with those of phentolamine, yohimbine and l‐arginine. The effects of sildenafil, phentolamine, yohimbine and l‐arginine were studied on the response of the RCC to electrical field stimulation (EFS) as well as on the phenylephrine (PE, 3 × 10−6 m)‐induced tone. EFS caused transient, frequency‐dependent relaxation of the RCC that was inhibited by the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine (3 × 10−5 m). Sildenafil (1 × 10−9–1 × 10−6 m) and phentolamine (1 × 10−9–1 × 10−6 m) enhanced the EFS‐induced relaxation in a concentration‐dependent manner with ED50 of 0.056 ± 0.004 and 0.572 ±0.035 μm at 8 Hz, respectively, yohimbine (3 × 10−7–3 × 10−5 m) and l‐arginine (3 × 10−6–3 × 10−4 m) did not show significant effects (ED50 at 8 Hz = 35.84 ±2.24 and 2.164 ± 0.174 μm, respectively). Sildenafil (1 × 10−9 and 1 × 10−8 m) potentiated the EFS‐induced relaxation caused by l‐arginine (3 × 10−5 m). Sildenafil, phentolamine, yohimbine and l‐arginine reduced the PE‐induced tone to different extents; the ED50 values were 0.81 ± 0.097, 0.49 ± 0.025 and 13.97 ± 1.10 μm, respectively. Maximum concentration of l‐arginine used failed to produce 50% relaxation (ED20 = 221.82 ± 15.71 μm). The muscle relaxant effects of different combinations of sildenafil and l‐arginine on PE‐induced tone did not differ significantly from the sum of the individual effects. The results demonstrate that sildenafil, when compared to other drugs used in penile erection dysfunction, shows the highest potency on the nitrergic transmission in the RCC. On the other hand, phentolamine was found to possess the highest potency in inducing relaxation of RCC proving that its action is independent on the stimulated neurogenic system. In addition, the combination of less effective doses of sildenafil and l‐arginine has a potential advantage on erectile functions. The importance of this combination remains to be solved clinically.

Evaluation of l-arginine on kidney function and vascular reactivity following ischemic injury in rats: Protective effects and potential interactions

BACKGROUND There is an interaction between many cell types involved in the pathophysiology of ischemic acute renal failure. Nitric oxide (NO) precursors, especially l-arginine, may have protective effects on tissue ischemia/reperfusion injury (IRI); however, their molecular mechanisms are unclear. In the present study, the interaction between l-arginine, cyclo-oxygenase (COX)-2 and reactive oxygen species (ROS) in the pathogenesis of ischemic acute renal failure was investigated. METHODS Ischemia/reperfusion injury model in rats was used and various biochemical parameters examined. The rat isolated aortic rings served as model for hypoxia/reoxygenation where endothelium dependent and independent relaxations were exerted. RESULTS Pre-treatment of rats subjected to IRI with l-arginine (125mg/kg) significantly reduced kidney MDA levels, elevated kidney SOD activity, GSH level and total NO levels at 24 and 48h after reperfusion. Kidney COX-2 level was only different in the l-arginine-treated group 48h after reperfusion compared to the IRI group. Pre-treatment with l-arginine (10(-2)M) alone or in combination with celecoxib significantly potentiated the acetylcholine (Ach)-induced relaxations in control and hypoxic rings. The effect of the combination was synergistic only in hypoxic rings. Addition of ascorbic acid to the celecoxib-arginine combination did not produce further potentiation. Sodium nitroprusside-induced relaxations in control and hypoxic rings were potentiated by l-arginine or celecoxib-arginine combination but not by ascorbic acid. CONCLUSIONS The protective effect of l-arginine may result from the interaction between NO and ROS and increased NO bioavailability. The protective effects of combined celecoxib and l-arginine against IRI could be attributed to their antioxidant activity which exceeded that of ascorbic acid.

Novel Water‐soluble Curcumin Derivative Mediating Erectile Signaling

INTRODUCTION Curcumin is an inducer of heme oxygenase enzyme-1 (HO-1) that is involved in erectile signaling via elevating cyclic guanosine monophosphate (cGMP)levels. AIM To assess the effect of oral administration of a water-soluble long-acting curcumin derivative on erectile signaling. METHODS Two hundred and thirty six male white albino rats were divided into four groups; group 1 (N = 20) includes control. Group 2 (N = 72) was equally divided into four subgroups; subgroup 1 received pure curcumin (10 mg/kg), subgroup 2 received the long-acting curcumin derivative (2 mg/kg), subgroup 3 received the long-acting curcumin derivative (10 mg/kg), and subgroup 4 received sildenafil (4 mg/kg). Subgroups were sacrificed after the first, second, and third hour. Group 3 (N = 72) was equally divided into the same four subgroups already mentioned and were sacrificed after 24 hours, 48 hours, and 1 week. Group 4 (N = 72) was subjected to intracavernosal pressure (ICP) measurements 1 hour following oral administration of the same previous doses in the same rat subgroups. MAIN OUTCOME MEASURE Cavernous tissue HO enzyme activity, cGMP, and ICP. RESULTS In group 2, there was a significant progressive maintained elevation of HO activity and cGMP tissue levels starting from the first hour in subgroups 3 and 4, whereas, the rise in HO activity and cGMP started from second hour regarding the other rat subgroups. Sildenafil effect decreased after 3 hours. In group 3, there was a significant maintained elevation of HO activity and cGMP tissue levels extended to 1 week as compared to controls for all rat subgroups that received both forms of curcumin. In group 4, long-acting curcumin derivative exhibited more significant potentiation of intracavernosal pressure as compared to control and to the pure curcumin. CONCLUSION Water-soluble long-acting curcumin derivative could mediate erectile function via upregulating cavernous tissue cGMP.

Effect of sildenafil on the isolated rat aortic rings

Sildenafil, a highly selective inhibitor of PDE 5, is effective in the treatment of erectile dysfunction. Penile erection involves relaxation of smooth muscle of corpus cavernosum and its associated arterioles. The objective of this study was to investigate the effect of sildenafil on nitric oxide/cyclic guanosine monophosphate (NO/cGMP)‐dependent relaxation of rat aortic rings. The contribution of sildenafil to the vasorelaxation of rat aortic rings was also investigated. Sildenafil produced significant potentiation of acetylcholine (ACh, 2 × 10−6 m)‐induced relaxation at concentration ≥1 × 10−8 m. Addition of sildenafil (1 × 10−7 m) to aortic rings failed to alter the effect of NG‐nitro‐l‐arginine (l‐NNA, 3 × 10−5 m) or methylene blue (MB, 3 × 10−5 m) on ACh response. Similarly, sildenafil (1 × 10−7 m) augmented significantly the vasorelaxation induced by sodium nitroprusside over the range of 1 × 10−9–1 × 10−8 m. When added to phenylephrine (3 × 10−6 m)‐precontracted rat aortic rings, sildenafil (1 × 10−9–1 × 10−4 m) induced concentration‐dependent relaxation reaching a maximum of 96.48 ± 1.44%. These relaxations were not significantly attenuated by previous incubation with l‐NNA (3 × 10−5 m) or MB (3 × 10−5 m). Denudation did not significantly affect the vasorelaxant effect of sildenafil. Sildenafil may act in the rat aortic rings through the amplification of NO/cGMP pathway. It may augment both basal endothelial NO function and exogenous NO‐dependent vasodilatation. However, sildenafil may act by a mechanism independent of NO/cGMP pathway and this mechanism contributes to its smooth muscle relaxant effect.