Suppression of TNBC metastasis by doxazosin, a novel dual inhibitor of c-MET/EGFR.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by aggressive growth and a high propensity for recurrence and metastasis. Simultaneous overexpression of c-MET and EGFR in TNBC is associated with worse clinicopathological features and unfavorable outcomes. Although the development of new c-MET inhibitors and the emergence of 3rd-generation EGFR inhibitors represent promising treatment options, the high costs involved limit the accessibility of these drugs. In the present study, we sought to investigate the therapeutic potential of doxazosin (DOXA), a generic drug for benign prostate hyperplasia, in targeting TNBC. METHODS: The effect of DOXA on TNBC cell lines in vitro was evaluated in terms of cell viability, apoptosis, c-MET/EGFR signaling pathway, molecular docking studies and impact on cancer stem cell (CSC)-like properties. An in vivo metastatic model with CSCs was used to evaluate the efficacy of DOXA. RESULTS: DOXA exhibits notable anti-proliferative effects on TNBC cells by inducing apoptosis via caspase activation. Molecular docking studies revealed the direct interaction of DOXA with the tyrosine kinase domains of c-MET and EGFR. Consequently, DOXA disrupts important survival pathways including AKT, MEK/ERK, and JAK/STAT3, while suppressing CSC-like characteristics including CD44high/CD24low subpopulations, aldehyde dehydrogenase 1 (ALDH1) activity and formation of mammospheres. DOXA administration was found to suppress tumor growth, intra- and peri-tumoral angiogenesis and distant metastasis in an orthotopic allograft model with CSC-enriched populations. Furthermore, no toxic effects of DOXA were observed in hepatic or renal function. CONCLUSIONS: Our findings highlight the potential of DOXA as a therapeutic option for metastatic TNBC, warranting further investigation.

Hemodynamic parameters in patients undergoing surgery for pheochromocytoma/paraganglioma: a retrospective study.

BACKGROUND: Pheochromocytoma (PHEO) and paraganglioma (PGL) are rare neuroendocrine tumors characterized by hemodynamic instability, caused by the paroxysmal release of catecholamines. Patients may develop cardiovascular complications in the perioperative phase due to the massive release of catecholamines, particularly during anesthetic induction and surgical manipulation of the tumor. The aim of this retrospective study was to evaluate the risk factors involved in perioperative hemodynamic instability in patients who underwent surgery for chromaffin tumors. METHODS: Forty patients (median age 55 [36.50-64.50]) undergone surgery for PHEO/abdominal PGL from January 2011 to December 2016 at the AOU Careggi (Florence, Italy) were retrospectively evaluated. Systolic, diastolic, and mean blood pressure were considered at baseline and during surgery. Patients with blood pressure steadily < 140/90 mmHg before surgery were considered "adequately prepared". A preoperative therapy with doxazosin, a selective alpha-1 blocker, was started in all patients for at least 14 days prior to the surgery. The presence of hemodynamic instability was reported. RESULTS: Comparing males and females, a significant difference in doxazosin daily dose (p = 0.018), systolic blood pressure (p = 0.048), and in the proportion of adequately prepared patients (p = 0.031) emerged. A positive correlation between preoperative daily dose of doxazosin, tumor size (B = 0.60, p < 0.001), and urinary normetanephrine levels (B = 0.64, p < 0.001) was also observed. Hemodynamic instability occurred in 30.0% of patients. The absence of adequate preparation (p = 0.012) before surgery, urinary normetanephrine levels (NMNur p = 0.039), and surgery time (minutes) (p = 0.021) resulted as risk factors of hemodynamic instability in our series. The use of intraoperative drugs was higher in patients with hemodynamic instability (p < 0.001). A pre-surgical SBP level of > 133 mmHg (OR = 6 CI95% 1.37-26.20, p = 0.017) and an intraoperative SBP and MBP levels of > 127 mmHg (OR = 28.80 CI95% 2.23-371.0, p = 0.010) and > 90 mmHg (OR = 18.90 CI95% 1.82-196.0, p = 0.014), respectively, were identified as effective thresholds to recognize patients at higher risk of HI. CONCLUSIONS: A preoperative therapy with alpha-blockers is useful, but not sufficient to avoid surgical risks. Patients with higher pre-surgical levels of NMNur, pre-surgical SBP > 133 mmHg, and/or intraoperative SBP > 127 mmHg and MBP > 90 mmHg, should be carefully monitored. A multidisciplinary approach is indispensable to optimize the management of PHEOs/abdominal PGLs in order to reduce surgical complications.

Drug repositioning: doxazosin attenuates the virulence factors and biofilm formation in Gram-negative bacteria.

The resistance development is an increasing global health risk that needs innovative solutions. Repurposing drugs to serve as anti-virulence agents is suggested as an advantageous strategy to diminish bacterial resistance development. Bacterial virulence is controlled by quorum sensing (QS) system that orchestrates the expression of biofilm formation, motility, and virulence factors production as enzymes and virulent pigments. Interfering with QS could lead to bacterial virulence mitigation without affecting bacterial growth that does not result in bacterial resistance development. This study investigated the probable anti-virulence and anti-QS activities of α-adrenoreceptor blocker doxazosin against Proteus mirabilis and Pseudomonas aeruginosa. Besides in silico study, in vitro and in vivo investigations were conducted to assess the doxazosin anti-virulence actions. Doxazosin significantly diminished the biofilm formation and release of QS-controlled Chromobacterium violaceum pigment and virulence factors in P. aeruginosa and P. mirabilis, and downregulated the QS encoding genes in P. aeruginosa. Virtually, doxazosin interfered with QS proteins, and in vivo protected mice against P. mirabilis and P. aeruginosa. The role of the membranal sensors as QseC and PmrA was recognized in enhancing the Gram-negative virulence. Doxazosin downregulated the membranal sensors PmR and QseC encoding genes and could in silico interfere with them. In conclusion, this study preliminary documents the probable anti-QS and anti-virulence activities of doxazosin, which indicate its possible application as an alternative or in addition to antibiotics. However, extended toxicological and pharmacological investigations are essential to approve the feasible clinical application of doxazosin as novel efficient anti-virulence agent. KEY POINTS: • Anti-hypertensive doxazosin acquires anti-quorum sensing activities • Doxazosin diminishes the virulence of Proteus mirabilis and Pseudomonas aeruginosa • Doxazosin could dimmish the bacterial espionage.

Anti-cell Proliferative Mechanism of Doxazosin on Human Oral Cancer Cells Through the Modulation of Antioxidant and Apoptotic Pathway.

Oral squamous cell carcinoma (OSCC), a global threatening disease, is reported mostly in the middle and elderly male population. Even though the exact cause of OSCC was not known, consumption of tobacco in any form has been reported in most of OSCC patients. OSCC is a massive invasive type of cancer which easily spreads to the distant organs. Hence treating it at appropriate time is necessary and the rate of OSCC incidence is also constantly increasing. At present, chemoradiation is the only therapy prescribed for OSCC patients which renders various side effects. Hence, the treatment with lesser side effect was of current research interest. Doxazosin (α1 adrenorecptor antagonist) had been proven to render anticancer effect in prostate, renal, hepatic, and ovarian cancers but its role in oral cancer cells was not been elucidated. Therefore, we have assessed the anticancer effect of doxazosin on oral squamous cancer cells via through the induction of apoptosis, and antioxidant property. The cytoprotective effect of doxazosin on normal Vero cells and anticancer effect on oral cancer KB cells were analyzed with MTT assay. Doxazosin antioxidant activity were analyzed by their reactivity with free radicals and metal ions by the method of FRAP, DPPH, chemilumiscence, and ORAC assay. The antioxidant levels were also assessed by TBARS, SOD, and glutathione levels, and later on apoptosis staining techniques like DCFH-DA, Rhodamine 123, and AO/EtBr stain were conducted. Apoptosis was confirmed by estimating the levels of apoptotic proteins in doxazosin-treated KB human oral cancer cells by ELISA method. The results from our study show that doxazosin is a potent antioxidant and it significantly induces apoptosis in human oral cancer by altering various cellular molecules at downstream signaling which has been depict in the results. Our study proves doxazosin as a potent anticancer drug which may be used in the treatment of oral carcinoma, if it is subjected to further research using human clinical trials.

Efficacy of Antihypertensive Drugs of Different Classes After Renal Denervation in Spontaneously Hypertensive Rats.

BACKGROUND: Renal denervation (RDN) lowers blood pressure (BP) in patients with uncontrolled hypertension. Limited data exist on the effectiveness of different antihypertensive medications following RDN on BP and maladaptive cardiac phenotypes. METHODS: Eighty-nine male spontaneously hypertensive rats with continuous BP recording underwent RDN or sham operation. Ten days postsurgery, spontaneously hypertensive rats were randomized to receive no antihypertensive medication, amlodipine, olmesartan, hydrochlorothiazide, bisoprolol, doxazosin, or moxonidine for 28 days. Cardiac remodeling was determined histologically, and activation of the renin-angiotensin-aldosterone system was explored. RESULTS: Before initiation of antihypertensive drugs, RDN reduced mean arterial pressure (-12.6 mm Hg [95% CI, -14.4 to -10.8]; P<0.001). At study end, mean arterial pressure was lower in RDN compared with sham operation in drug-naïve controls (P=0.006), olmesartan (P=0.002), amlodipine (P=0.0004), hydrochlorothiazide (P=0.006), doxazosin (P=0.001), and bisoprolol (P=0.039) but not in animals receiving moxonidine (P=0.122). Compared with pooled BP change of all other drug classes, mean arterial pressure change was largest for olmesartan (-15.9 mm Hg [95% CI, -18.6 to -13.2]; P<0.001) and amlodipine (-12.0 mm Hg [95% CI, -14.7 to -9.3]; P<0.001). In drug-naïve controls, RDN reduced plasma renin activity (-5.6%¸ P=0.03) and aldosterone concentration (-53.0%; P=0.005). In the presence of antihypertensive medication, plasma renin activity and aldosterone remained unchanged after RDN. Cardiac remodeling was not affected by RDN alone. In animals receiving olmesartan after RDN, cardiac perivascular fibrosis was attenuated. Amlodipine and bisoprolol following RDN reduced cardiomyocyte diameter. CONCLUSIONS: Following RDN, treatment with amlodipine and olmesartan resulted in the largest BP reduction. Antihypertensive medications mediated heterogeneous effects on renin-angiotensin-aldosterone system activity and cardiac remodeling.

6-nitrodopamine is a major endogenous modulator of human vas deferens contractility.

BACKGROUND: Rat isolated vas deferens releases 6-nitrodopamine (6-ND), and the spasmogenic activity of this novel catecholamine is significantly reduced by tricyclic compounds such as amitriptyline, desipramine, and carbamazepine and by antagonists of the α1 -adrenergic receptors such as doxazosin, tamsulosin, and prazosin. OBJECTIVES: To investigate the liberation of 6-ND by human epididymal vas deferens (HEVDs) and its pharmacological actions. METHODS: The in vitro liberation of 6-ND, dopamine, noradrenaline, and adrenaline from human vas deferens was evaluated by LC-MS/MS. The contractile effect of the catecholamines in HEVDs was investigated in vitro. The action of tricyclic antidepressants was evaluated on the spasmogenic activity ellicited by the catecholamines and by the electric-field stimulation (EFS). The tissue was also incubated with the inhibitor of nitric oxide (NO) synthase L-NAME and the release of catecholamines and the contractile response to EFS were assessed. RESULTS: 6-ND is the major catecholamine released from human vas deferens and its synthesis/release is inhibited by NO inhibition. The spasmogenic activity elicited by EFS in the human vas deferens was blocked by tricyclic antidepressants only at concentrations that selectively antagonize 6-ND induced contractions of the human vas deferens, without affecting the spasmogenic activity induced by dopamine, noradrenaline, and adrenaline in this tissue. Incubation of the vas deferens with L-NAME reduced both the 6-ND release and the contractions induced by EFS. DISCUSSION AND CONCLUSION: 6-ND should be considered a major endogenous modulator of human vas deferens contractility and possibly plays a pivotal role in the emission process of ejaculation. It offers a novel and shared mechanism of action for tricyclic antidepressants and α1 -adrenergic receptor antagonists.

Comparison of Preoperative Alpha-blockade for Resection of Paraganglioma and Pheochromocytoma.

OBJECTIVE: Phenoxybenzamine (nonselective, noncompetitive alpha-blocker) is the preferred drug for preoperative treatment of pheochromocytoma, but doxazosin (selective, competitive alpha-blocker) may be equally effective. We compared the efficacy of doxazosin vs phenoxybenzamine. METHODS: We conducted a prospective study of patients undergoing pheochromocytoma or paraganglioma resection by randomizing pretreatment with phenoxybenzamine or doxazosin at a single tertiary referral center. The high cost of phenoxybenzamine led to high crossover to doxazosin. Randomization was halted, and a consecutive historical cohort of phenoxybenzamine patients was included for a case-control study design. The efficacy of alpha-blockade was assessed with preinduction infusion of incremental doses of phenylephrine. The primary outcomes were mortality, cardiovascular complications, and intensive care unit admission. The secondary outcomes were hemodynamic instability index (proportion of operation outside of hemodynamic goals), adequacy of blockade by the phenylephrine titration test, and drug costs. RESULTS: Twenty-four patients were prospectively enrolled (doxazosin, n = 20; phenoxybenzamine, n = 4), and 15 historical patients treated with phenoxybenzamine were added (total phenoxybenzamine, n = 19). No major cardiovascular complications occurred in either group. The phenylephrine dose-response curves showed less blood pressure rise in the phenoxybenzamine than in the doxazosin group (linear regression coefficient = 0.008 vs 0.018, P = .01), suggesting better alpha-blockade in the phenoxybenzamine group. The median hemodynamic instability index was 14% vs 13% in the phenoxybenzamine and doxazosin groups, respectively (P = .56). The median highest daily cost of phenoxybenzamine was $442.20 compared to $5.06 for doxazosin. CONCLUSION: Phenoxybenzamine may blunt intraoperative hypertension better than doxazosin, but this difference did not translate to fewer cardiovascular complications and is offset by a considerably increased cost.

α-1 Adrenergic receptor antagonist doxazosin reverses hepatic stellate cells activation via induction of senescence.

BACKGROUND: Activated hepatic stellate cells (aHSCs) are the main effector cells during liver fibrogenesis. α-1 adrenergic antagonist doxazosin (DX) was shown to be anti-fibrotic in an in vivo model of liver fibrosis (LF), but the mechanism remains to be elucidated. Recent studies suggest that reversion of LF can be achieved by inducing cellular senescence characterized by irreversible cell-cycle arrest and acquisition of the senescence-associated secretory phenotype (SASP). AIM: To elucidate the mechanism of the anti-fibrotic effect of DX and determine whether it induces senescence. METHODS: Primary culture-activated rat HSCs were used. mRNA and protein expression were measured by qPCR and Western blot, respectively. Cell proliferation was assessed by BrdU incorporation and xCelligence analysis. TGF-ß was used for maximal HSC activation. Norepinephrine (NE), PMA and m-3M3FBS were used to activate alpha-1 adrenergic signaling. RESULTS: Expression of Col1α1 was significantly decreased by DX (10 µmol/L) at mRNA (-30 %) and protein level (-50 %) in TGF-ß treated aHSCs. DX significantly reduced aHSCs proliferation and increased expression of senescence and SASP markers. PMA and m-3M3FBS reversed the effect of DX on senescence markers. CONCLUSION: Doxazosin reverses the fibrogenic phenotype of aHSCs and induces the senescence phenotype.

Doxazosin Attenuates Liver Fibrosis by Inhibiting Autophagy in Hepatic Stellate Cells via Activation of the PI3K/Akt/mTOR Signaling Pathway.

PURPOSE: To investigate the effect of doxazosin on autophagy and the activation of hepatic stellate cells (HSCs) in vivo and in vitro and determine the underlying mechanism. METHODS: In vivo, a mouse liver fibrosis model was induced by the intraperitoneal injection of carbon tetrachloride (CCl4). Doxazosin was administered at doses of 2.5, 5 and 10 mg/(kg*day) by gavage. After 20 weeks, blood and liver tissues were collected for serological and histological analysis, respectively. Blood analysis, hematoxylin and eosin (HE) staining, Masson's trichrome staining, immunohistochemistry and immunofluorescence staining were used to measure the extent of liver fibrosis in model and control mice. In vitro, the human HSC cell line LX-2 was cultured and treated with different doses of doxazosin for the indicated times. The effects of doxazosin on LX-2 cell proliferation and migration were examined by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The number of autophagosomes in LX-2 cells was observed by transmission electron microscopy (TEM). Infection with green fluorescent protein (GFP)-LC3B adenovirus, GFP-red fluorescent protein (RFP)-LC3B adenovirus and mCherry-EGFP-LC3 adeno-associated virus was performed to examine changes in autophagic flux in vitro and in vivo. Cell apoptosis was measured by flow cytometry in vitro and by TUNEL assays both in vitro and in vivo. Immunoblotting was performed to evaluate the expression levels of proteins related to fibrosis, autophagy, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR). RESULTS: Doxazosin inhibited HSC proliferation and migration. HSC activation was attenuated by doxazosin in a concentration-dependent manner in vivo and in vitro. Doxazosin also blocked autophagic flux and induced apoptosis in HSCs. In addition, the PI3K/Akt/mTOR pathway was activated by doxazosin and regulated fibrosis, autophagy and apoptosis in HSCs. CONCLUSION: The study confirmed that doxazosin could inhibit autophagy by activating the PI3K/Akt/mTOR signaling pathway and attenuate liver fibrosis.

Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth.

EphA2 receptor kinase could become a novel target for anti-glioblastoma treatment. Doxazosin previously identified acts like the endogenous ligand of EphA2 and induces cell apoptosis. Through lead structure modification a derivative of Doxazosin possessing unique dimeric structure showed an improvement in the activity. In the current study, we expanded the dimeric scaffold by lead optimization to explore the chemical space of the conjoining moieties and a slight variation to the core structure. 27 new derivatives were synthesized and examined with EphA2 overexpressed and wild type glioblastoma cell lines for cell proliferation and EphA2 activation. Three new compounds 3d, 3e, and 7bg showed potent and selective activities against the growth of EphA2 overexpressed glioblastoma cells. Dimer 3d modification replaces the long alkyl chain with a short polyethylene glycol chain. Dimer 7bg has a relatively longer polyethylene glycol chain in comparison to compound 3d and the length is more similar to the lead compound. Whereas dimer 3e has a rigid aromatic linker exploring the chemical space. The diversity of the linkers in the active suggest additional hydrogen binding sites has a positive correlation to the activity. All three dimers showed selective activity in EphA2 overexpressed cells, indicating the activity is correlated to the EphA2 targeting effect.

Evaluation of the effect of doxasozin and zonisamide on voluntary ethanol intake in mice that experienced chronic intermittent ethanol exposure and stress.

The comorbidity between alcohol use disorder and post-traumatic stress disorder represents a serious health care burden with few effective treatment options. The current study was designed to evaluate the effect of an alpha 1 receptor antagonist (doxazosin) and a novel anticonvulsant (zonisamide) in a model of alcohol (ethanol) dependence and stress exposure. The main dependent variable was voluntary ethanol intake in mice that experienced chronic intermittent ethanol (CIE) exposure and forced swim stress (FSS) alone, and in combination. Adult male and female C57BL/6J mice had access to a single bottle of 15% (v/v) ethanol for 1-hr in the home cage, 3-hr into the dark phase of the light/dark cycle. Once stable ethanol intake was established (~4 weeks), mice were separated into four groups (CTL, CIE, FSS, CIE + FSS). Mice in the FSS condition received 10-min FSS exposure 4-hr prior to drinking sessions (remaining mice were not disturbed). During baseline and the first two test cycles, all mice received vehicle (saline) injections (IP) 30-min before ethanol access. As previously observed, FSS increased ethanol drinking in dependent (CIE-exposed) mice but not in nondependent control (CTL) mice. In the following test cycles mice were evaluated for ethanol intake after administration of doxazosin, zonisamide or their combination. Results indicated that the three doses of doxazosin evaluated significantly reduced voluntary ethanol intake in all mice. Zonisamide had a more modest effect and may require a more prolonged treatment regime. The combined administration of both compounds was not more effective than each drug alone. This study suggests that doxazosin is reliable at reducing voluntary ethanol intake in mice independently of their history of ethanol dependence and stress exposure.

Potential role of EphrinA2 receptors in postconditioning induced cardioprotection in rats.

EphA2 receptor has emerged as a novel cardioprotective target against myocardial infarction by preserving cardiac function, limiting infarct size and inflammation and enhancing cell survival via elevating phosphorylated Akt protein levels. However, the role of Eph receptors in postconditioning remains to be elucidated. Thus, the present study was designed to explore the role of EphA2 receptors in cardioprotective mechanism of postconditioning by employing Doxazosin as EphA2 receptor agonist, Lithocholic acid as antagonist and Wortmannin as specific phosphoinositide 3-kinase (PI3K) inhibitor. In Langendorff perfused isolated rat hearts, exposure of ischemia for 30 min succeeded by reperfusion for 2 h produced cardiac damage as determined by increase in size of infarct, LVDP, liberation of LDH and CK in effluent from coronary arteries. The reperfused hearts were homogenized and tissue concentrations of TBARs, reduced GSH and Catalase were determined. A marked rise in infarct size, liberation of LDH and CK in effluent and TBARs in myocardial tissue was observed in ischemic and reperfused hearts. Ischemic postconditioning comprising of 6 alternate episodes of 10 s ischemia and 10 s reperfusion and pharmacological post-conditioning by Doxazosin infusion for 5 min Before reperfusion confers significant protection against myocardial injury as manifested by remarkably decreased infarct size, levels of LDH, CK and tissue TBARs along with increase in GSH and Catalase activity. Pre-treatment of EphA2 antagonist, Lithocholic acid and PI3K inhibitor, Wortmannin attenuated the cardioprotective effect of postconditioning. Our results suggest that EphA2 receptors may be involved in postconditioning mediated cardioprotection probably through PI3K/Akt pathway.

Norepinephrine Inhibits the Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells via ß2-Adrenoceptor-Mediated ERK1/2 and PKA Phosphorylation.

Bone marrow-derived mesenchymal stem cells (BMSCs) represent an alternative to chondrocytes to support cartilage regeneration in osteoarthritis (OA). The sympathetic neurotransmitter norepinephrine (NE) has been shown to inhibit their chondrogenic potential; however, their proliferation capacity under NE influence has not been studied yet. Therefore, we used BMSCs obtained from trauma and OA donors and compared the expression of adrenergic receptors (AR). Then, BMSCs from both donor groups were treated with NE, as well as with combinations of NE and α1-, α2- or ß1/2-AR antagonists (doxazosin, yohimbine or propranolol). Activation of AR-coupled signaling was investigated by analyzing ERK1/2 and protein kinase A (PKA) phosphorylation. A similar but not identical subset of ARs was expressed in trauma (α2B-, α2C- and ß2-AR) and OA BMSCs (α2A-, α2B-, and ß2-AR). NE in high concentrations inhibited the proliferation of both trauma and OA BMCSs significantly. NE in low concentrations did not influence proliferation. ERK1/2 as well as PKA were activated after NE treatment in both BMSC types. These effects were abolished only by propranolol. Our results demonstrate that NE inhibits the proliferation and accordingly lowers the regenerative capacity of human BMSCs likely via ß2-AR-mediated ERK1/2 and PKA phosphorylation. Therefore, targeting ß2-AR-signaling might provide novel OA therapeutic options.

Polycomb-mediated repression of EphrinA5 promotes growth and invasion of glioblastoma.

Glioblastoma (GBM) is the most common and most aggressive intrinsic brain tumour in adults. Integrated transcriptomic and epigenomic analyses of glioblastoma initiating cells (GIC) in a mouse model uncovered a novel epigenetic regulation of EfnA5. In this model, Bmi1 enhances H3K27me3 at the EfnA5 locus and reinforces repression of selected target genes in a cellular context-dependent fashion. EfnA5 mediates Bmi1-dependent proliferation and invasion in vitro and tumour formation in an allograft model. Importantly, we show that this novel Polycomb feed-forward loop is also active in human GIC and we provide pre-clinical evidence of druggability of the EFNA5 signalling pathway in GBM xenografts overexpressing Bmi1.

Doxazosin down-regulates sodium-glucose cotransporter-2 and exerts a renoprotective effect in rat models of acute renal injury.

Sodium-glucose cotransporter-2 (SGLT2) is known to be involved in the progression of acute renal injury (ARI) and is regulated by different mediators in the kidneys including extracellular signal-regulated kinase (ERK), hypoxia-inducible factor 1 alpha (HIF1α) and prostaglandin E2 (PGE2). In the present study, we investigated the possible protective effect of doxazosin on renal ischaemia/reperfusion (IR) and glycerol-induced ARI by determining its effect on SGLT2 via modifying ERK-HIF1α pathway and/or PGE2. Rats were divided into control, sham or IR where the rats received the vehicle, doxazosin (8 mg/kg) or the SGLT2 inhibitor, dapagliflozin (10 mg/kg) for 3 days followed by 45 minutes bilateral renal ischaemia then 24 hours reperfusion. Another group of rats received the vehicle, doxazosin or dapagliflozin for three days followed by injection of 50% glycerol (8 mL/kg, IM) or saline. Kidney function tests, systolic blood pressure (SBP), oxidative stress markers (malondialdehyde [MDA] and NADPH oxidase), nitric oxide (NO), inducible nitric oxide synthase (iNOS), HIF1α, ERK phosphorylation and PGE2 levels were determined. Additionally, renal sections were used for immunological expression of SGLT2. ARI rats showed significantly increased SBP; worsened kidney function tests; increased oxidative stress, iNOS, NO, HIF1α levels; and decreased PGE2 and ERK phosphorylation along with up-regulated SGLT2. Doxazosin treatment protected against the kidney damage and attenuated the associated biochemical changes. Doxazosin has a direct renoprotective effect possibly by down-regulating SGLT2.

Revisiting the Pharmacodynamic Uroselectivity of α 1-Adrenergic Receptor Antagonists.

α1-Adrenoceptor (AR) antagonists are widely used for the relief of urinary retention secondary to benign prostatic hyperplasia (BPH). While the five Food and Drug Administration-approved α 1-AR antagonists (terazosin, doxazosin, alfuzosin, tamsulosin, and silodosin) share similar efficacy, they differ in tolerability, with reports of ejaculatory dysfunction. The aim of the present work was to revisit their α 1-AR subtype selectivity as well as of LDT5 (1-(2-methoxyphenyl)-4-[2-(3,4-dimethoxyphenyl) ethyl]piperazine monohydrochloride), a compound previously described as a multitarget antagonist of α 1A-/α 1D-AR and 5-HT1A receptors, and to estimate their affinity for D2, D3, and 5-HT1A receptors, which are putatively involved in ejaculatory dysfunction. Competition binding assays were performed with native (D2, 5-HT1A) or transfected (human α 1A-, α 1B-, α 1Dt-AR, and D3) receptors for determination of the drug's affinities. Tamsulosin and silodosin have the highest affinities for α 1A-AR, but only silodosin is clearly a selective α 1A-AR antagonist, with K i ratios of 25.3 and 50.2 for the α 1D- and α 1B-AR, respectively. Tamsulosin, silodosin, and LDT5 (but not terazosin, doxazosin, and alfuzosin) have high affinity for the 5-HT1A receptor (K i around 5-10 nM), behaving as antagonists. We conclude that the uroselectivity of tamsulosin is not explained by its too-low selectivity for the α 1A- versus α 1B-AR, and that its affinity for D2 and D3 receptors is probably too low for explaining the ejaculatory dysfunction reported for this drug. Present data also support the design of "better-than-LDT5" new multitarget lead compounds with pharmacokinetic selectivity based on poor brain penetration and that could prevent hyperplastic cell proliferation and BPH progression. SIGNIFICANCE STATEMENT: The present work revisits the uroselectivity of the five Food and Drug Administration-approved α1 adrenoceptor antagonists for the treatment of benign prostatic hyperplasia (BPH). Contrary to what has been claimed by some, our results indicate that the uroselectivity of tamsulosin is probably not fully explained by its too-weak selectivity for the α1A versus α1B adrenoceptors. We also show that tamsulosin affinity for D3 and 5-HT1A receptors is probably too low for explaining the ejaculatory dysfunction reported for this drug. Based on our lead compound LDT5, present data support the search for a multitarget antagonist of α1A-α1D and 5-HT1A receptors with poor brain penetration as an alternative for BPH treatment.

Effects of doxazosin mesylate versus nifedipine on blood pressure variability in hypertensive patients: a randomized crossover study (SIMILAR).

OBJECTIVE: Blood pressure variability (BPV) is a powerful predictor of end-organ damage, cardiovascular events and mortality independently of the BP level. Calcium channel blockers may offer an advantage over other first-line antihypertensive drugs by preventing increased BPV. But the effect of alpha-receptor blockers on BPV in hypertensive patients is still unclear. METHODS: In this crossover trial, 36 hypertensive patients were randomly assigned to two groups, receiving doxazosin mesylate gastrointestinal therapeutic system (GITS) (4 mg/day) or nifedipine GITS (30 mg/day) for 12 weeks, followed by a 2-week washout period then a 12-week crossover phase. At baseline and after 12-week treatment, 24-hour ambulatory BP monitoring was performed. BPV was evaluated through standard deviation (SD), coefficient of variation (CV), and average real variability (ARV) of systolic BP (SBP) and diastolic BP (DBP) during daytime, nighttime and over 24 hours. RESULTS: After 12-week treatment, both doxazosin and nifedipine significantly decreased SBP and DBP (P < 0.05), whereas no between-group differences were shown (P>0.05). Systolic BPV (24-hour SD, CV, and ARV; daytime SD; nighttime SD and CV) and diastolic BPV (24-hour SD and ARV) were significantly lowered by nifedipine (P < 0.05); doxazosin resulted in significant reductions of systolic BPV (24-hour SD, CV and ARV; daytime SD; nighttime SD) and diastolic BPV (nighttime SD and CV) (P < 0.05). Doxazosin was revealed to be as effective as nifedipine for reducing BPV (P > 0.05) except for 24-hour SBP ARV. CONCLUSIONS: Doxazosin mesylate GITS had similar therapeutic effects on BP, BP SD, and BP CV lowering as nifedipine GITS in patients with mild-to-moderate essential hypertension.

Alpha-Blockers As Colorectal Cancer Chemopreventive: Findings from a Case-Control Study, Human Cell Cultures, and In Vivo Preclinical Testing.

A retrospective case-controlled analysis was performed to identify drug candidates in the current use that may prevent colorectal cancer, outside of aspirin. A total of 37,510 patients aged ≥20 years were assessed to identify subjects who had been diagnosed with colorectal cancer by colonoscopy without a previous diagnosis of colorectal cancer, inflammatory bowel disease (IBD), or gastrointestinal symptoms; 1,560 patients were identified who were diagnosed with colorectal cancer by colonoscopy. The patients with colorectal cancer were matched with 1,560 age, gender, family history of colorectal cancer and comorbidity-matched control patients who were not diagnosed with colorectal cancer at colonoscopy. The medication histories were compared between the two groups. Next, candidate drugs that were more frequently used by the control patients were selected and their effects on human colorectal cancer cell lines in vitro and an inflammation-induced mouse model of colorectal cancer were tested. Putative colorectal cancer preventative agents were identified, including aspirin, vitamin D, vitamin B, vitamin C, vitamin E, xanthine oxidase inhibitor, alpha-blockers, angiotensin receptor blocker, nateglinide, probiotics, thienopyridine, folic acid, nitrovasodilators, bisphosphonates, calcium channel blockers, steroids, and statins (P < 0.05). Alpha-blockers and xanthine oxidase inhibitors were selected for further study because these agents have not been analyzed previously as factors that may affect colorectal cancer outcomes. In vitro doxazosin (alpha-blocker), but not febuxostat (xanthine oxidase inhibitor), suppressed the proliferation of human colorectal cancer cells. Doxazosin also decreased tumorigenesis in an AOM/DSS mouse colorectal cancer model. Alpha-blockers may prevent colorectal cancer.

Dual Effect of Doxazosin: Anticancer Activity on SH-SY5Y Neuroblastoma Cells and Neuroprotection on an In Vitro Model of Alzheimer's Disease.

Several studies have demonstrated the antitumor effect of doxazosin, an α1-adrenergic blocker, against glioma and breast, bladder and prostate cancers. Doxazosin is also being evaluated as a treatment for posttraumatic stress disorder (PTSD) and alcoholism, and α1-adrenergic blockers have been linked to neuroprotection in neurodegenerative disorders, such as Alzheimer's Disease (AD). Cancer and AD have an inverse relationship in many aspects, with several factors that contribute to apoptosis inhibition and proliferation being increased in cancers but decreased in AD. Neuroblastoma (NB) is a pediatric tumor derived from embryonic neural-crest cells, with an overall cure rate of 40%, despite aggressive treatment. Thus, due to the need of new therapeutic strategies against NB and neurodegenerative disorders and the inverse relationship between these diseases, we investigated whether doxazosin may serve as an antitumor and neuroprotective agent. We analyzed the drug's effects on undifferentiated and retinoic acid-differentiated SH-SY5Y human NB cells and on an in vitro model of organotypic hippocampal cultures exposed to amyloid-ß. Doxazosin showed antitumor effect on undifferentiated NB cells by induction of apoptosis, necrosis, cell cycle arrest and decrease of p-EGFRTyr1048 levels. On differentiated cells, doxazosin was less cytotoxic and increased p-EGFRTyr1048, p-AktSer473 and p-GSK-3ßSer9 levels. Moreover, the drug was able to protect hippocampal slices from amyloid-ß toxicity through prevention of GSK-3ß activation and of Tau hyperphosphorylation. Therefore, our results show that doxazosin has antitumor activity against undifferentiated NB and is neuroprotective on an in vitro model of Alzheimer's disease.

Modulation of α-adrenoceptor signalling protects photoreceptors after retinal detachment by inhibiting oxidative stress and inflammation.

BACKGROUND AND PURPOSE: Currently available treatments do not halt progression of photoreceptor death and subsequent visual impairment related to retinal detachment (RD) which is observed in various retinal disorders. This study investigated the neuroprotective effects of two adrenoceptor ligands, the α1 -adrenoceptor antagonist doxazosin and the α2 -adrenoceptor agonist guanabenz, against photoreceptor cell death in RD. EXPERIMENTAL APPROACH: We used a model of experimental RD in Brown-Norway rats induced by subretinal injection of sodium hyaluronate. Oxidative stress biomarkers and cytokine production were quantified with elisa. Protein expression levels and immunofluorescent labelling were determined in rats with RD and controls for mechanistic elucidation. The effects of systemic (i.p.) administration of doxazosin or guanabenz on photoreceptor apoptosis, retinal histology and electroretinography were evaluated in rats with RD and compared to the effects in vehicle controls. KEY RESULTS: Photoreceptors were the major source of RD-induced ROS overproduction in the rat retina through the regulation of NADPH oxidase. Systemic administration of doxazosin or guanabenz decreased the RD-induced production of ROS and proinflammatory cytokines, including IL-1ß and the chemokine CCL2, and suppressed retinal gliosis, resulting in attenuation of photoreceptor death and preservation of retinal structures and functions in RD. CONCLUSIONS AND IMPLICATIONS: Our findings point to α-adrenoceptors as novel therapeutic targets to provide photoreceptor protection and suggest that both doxazosin and guanabenz, two FDA-approved drugs, could be further explored to treat retinal diseases.