In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts.

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7-9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats.

BACKGROUND: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX. METHODS: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis. RESULTS: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX. CONCLUSIONS: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

Graft Preservation Solution DuraGraft® Alleviates Vascular Dysfunction Following In Vitro Ischemia/Reperfusion Injury in Rats.

Vascular ischemia/reperfusion injury (IRI) in patients undergoing coronary artery bypass grafting can result in graft failure and the need for repeat revascularization procedures. DuraGraft® has been shown to protect structure and function in saphenous vein grafts against IRI. We compared the effect of DuraGraft® to saline solution on arterial grafts submitted to IRI. Rat thoracic aortic rings were harvested and immediately mounted in organ bath chambers (control, n = 7 rats) or underwent cold ischemic preservation either in saline (IR, n = 9 rats) or DuraGraft® (IR+Dura, n = 9 rats). Vascular function was measured ex vivo and immunohistochemistry was performed. Impaired maximum vasorelaxation (Rmax) to ACh in the IR-group compared to controls was ameliorated by DuraGraft®, indicating an improvement in endothelial function (Rmax to ACh (%): IR + Dura 73 ± 2 vs. IR 48 ± 3, p < 0.05). Additionally, decreased aortic ring sensitivity to ACh (pD2-value: -log 50% maximum response) seen after IR in the saline group was increased by DuraGraft® (pD2 to ACh: IR+Dura 7.1 ± 0.1 vs. IR 6.3 ± 0.2, p < 0.05). Impaired maximum contractile response to phenylephrine and high potassium chloride concentrations in the IR group compared to controls was significantly improved by DuraGraft®. DuraGraft® alleviates vascular dysfunction following IRI by reducing nitro-oxidative stress and the expression of ICAM-1, without leukocytes engagement.

Reconditioning of circulatory death hearts by ex-vivo machine perfusion with a novel HTK-N preservation solution.

BACKGROUND: Warm ischemia followed by blood reperfusion is associated with reduced myocardial contractility. Circulatory death (CD) hearts are maintained by machine perfusion (MP) with blood. However, the impact of MP with histidine-tryptophane-ketoglutarate (HTK) or novel HTK-N solution on reconditioning of CD-heart contractility is unknown. METHODS: In a porcine model, native hearts were directly harvested (control), or CD was induced before harvesting, followed by left ventricular (LV) contractile assessment. In MP-groups, CD-hearts were maintained for 4 h by MP with blood (CD-B), cold oxygenated HTK (CD-HTK) or HTK-N (CD-HTK-N) before contractile evaluation (all groups n = 8). We performed immunohistochemistry of LV myocardial samples. We profiled myocardial expression of 84 oxidative stress-related genes and correlated the findings with myocardial contractility via a machine learning algorithm. RESULTS: HTK-N improved end-systolic pressure (ESP=172±10 vs 132±5 mmHg, p = 0.02) and maximal slope of pressure increment (dp/dtmax=2161±214 vs 1240±167 mmHg/s, p = 0.005) compared to CD, whereas CD-B failed to improve contractility. Dp/dtmax (2161±214 vs 1177±156, p = 0.08) and maximal rate of pressure decrement (dp/dtmin=-1501±228 vs -637±79, p = 0.005) were also superior in CD-HTK-N compared to CD-B. In CD-HTK-N, myocardial 4-hydroxynonenal (marker for oxidative stress; p<0.001), nitrotyrosine (marker for nitrosative stress; p = 0.004), poly(adenosine diphosphate-ribose)polymerase (marker for necrosis; p = 0.028) immunoreactivity and cell swelling (p = 0.008) were decreased compared to CD-B. Strong correlation of gene expression with ESP was identified for oxidative stress defense genes in CD-HTK-N. CONCLUSION: During harvesting procedure, MP with HTK-N reconditions CD-heart systolic and diastolic function by reducing oxidative and nitrosative stress and preventing cardiomyocytes from cell swelling and necrosis.

The Sodium-Glucose Cotransporter-2 Inhibitor Canagliflozin Alleviates Endothelial Dysfunction Following In Vitro Vascular Ischemia/Reperfusion Injury in Rats.

Vascular ischemia/reperfusion injury (IRI) contributes to graft failure and adverse clinical outcomes following coronary artery bypass grafting. Sodium-glucose-cotransporter (SGLT)-2-inhibitors have been shown to protect against myocardial IRI, irrespective of diabetes. We hypothesized that adding canagliflozin (CANA) (an SGLT-2-inhibitor) to saline protects vascular grafts from IRI. Aortic rings from non-diabetic rats were isolated and immediately mounted in organ bath chambers (control, n = 9-10 rats) or underwent cold ischemic preservation in saline, supplemented either with a DMSO vehicle (IR, n = 8-10 rats) or 50µM CANA (IR + CANA, n = 9-11 rats). Vascular function was measured, the expression of 88 genes using PCR-array was analyzed, and feature selection using machine learning was applied. Impaired maximal vasorelaxation to acetylcholine in the IR-group compared to controls was significantly ameliorated by CANA (IR 31.7 ± 3.2% vs. IR + CANA 51.9 ± 2.5%, p < 0.05). IR altered the expression of 17 genes. Ccl2, Ccl3, Ccl4, CxCr4, Fos, Icam1, Il10, Il1a and Il1b have been found to have the highest interaction. Compared to controls, IR significantly upregulated the mRNA expressions of Il1a and Il6, which were reduced by 1.5- and 1.75-fold with CANA, respectively. CANA significantly prevented the upregulation of Cd40, downregulated NoxO1 gene expression, decreased ICAM-1 and nitrotyrosine, and increased PECAM-1 immunoreactivity. CANA alleviates endothelial dysfunction following IRI.

Prospective multicentric validation of a novel prediction model for paroxysmal atrial fibrillation.

BACKGROUND: The early recognition of paroxysmal atrial fibrillation (pAF) is a major clinical challenge for preventing thromboembolic events. In this prospective and multicentric study we evaluated prediction scores for the presence of pAF, calculated from non-invasive medical history and echocardiographic parameters, in patients with unknown AF status. METHODS: The 12-parameter score with parameters age, LA diameter, aortic root diameter, LV,ESD, TDI A', heart frequency, sleep apnea, hyperlipidemia, type II diabetes, smoker, ß-blocker, catheter ablation, and the 4-parameter score with parameters age, LA diameter, aortic root diameter and TDI A' were tested. Presence of pAF was verified by continuous electrocardiogram (ECG) monitoring for up to 21 days in 305 patients. RESULTS: The 12-parameter score correctly predicted pAF in all 34 patients, in which pAF was newly detected by ECG monitoring. The 12- and 4-parameter scores showed sensitivities of 100% and 82% (95%-CI 65%, 93%), specificities of 75% (95%-CI 70%, 80%) and 67% (95%-CI 61%, 73%), and areas under the receiver operating characteristic (ROC) curves of 0.84 (95%-CI 0.80, 0.88) and 0.81 (95%-CI 0.74, 0.87). Furthermore, properties of AF episodes and durations of ECG monitoring necessary to detect pAF were analysed. CONCLUSIONS: The prediction scores adequately detected pAF using variables readily available during routine cardiac assessment and echocardiography. The model scores, denoted as ECHO-AF scores, represent simple, highly sensitive and non-invasive tools for detecting pAF that can be easily implemented in the clinical practice and might serve as screening test to initiate further diagnostic investigations for validating the presence of pAF. Prospective validation of a novel prediction model for paroxysmal atrial fibrillation based on echocardiography and medical history parameters by long-term Holter ECG.

Downregulation of HER3 by a novel antisense oligonucleotide, EZN-3920, improves the antitumor activity of EGFR and HER2 tyrosine kinase inhibitors in animal models.

Among the four human EGF receptor (HER) family members (EGFR, HER2, HER3, HER4), HER3 is of particular interest as it interacts with HER2 and EGFR via heterodimerization and is a key link to the phosphoinositide 3-kinase (PI3K)/AKT signal transduction axis. Recent studies indicate that HER3 plays a critical role in mediating resistance to agents that target EGFR or HER2. As HER3 lacks significant kinase activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors of HER3 have been sought as cancer therapeutics. We describe here a locked nucleic acid (LNA)-based HER3 antisense oligonucleotide, EZN-3920, that specifically downmodulated the expression of HER3, which was associated with growth inhibition. EZN-3920 effectively downmodulated HER3 expression, HER3-driven PI3K/AKT signaling pathway, and growth in tumors derived from BT474M1 breast and HCC827 lung carcinoma cell lines, which overexpress HER2 and EGFR, respectively. Furthermore, when EZN-3920 was coadministered with gefitinib or lapatinib in xenograft tumor models, enhanced antitumor activity compared with the effect of monotherapy was found. The effect was associated with a blockade of induced HER3 mRNA expression caused by lapatinib or gefitinib treatment. Finally, EZN-3920 sustained its antiproliferative effect in trastuzumab-resistant cells and three independently derived gefitinib-resistant cells. Our findings show that downmodulation of HER3 by EZN-3920 leads to the suppression of tumor growth in vitro and in vivo, suggesting that HER3 can be an effective target for the treatment of various cancers that have been activated by HER3 alone or where HER3 activation is associated with EGFR or HER2 expression.

Reduced expression of the androgen receptor by third generation of antisense shows antitumor activity in models of prostate cancer.

The androgen receptor (AR) is a member of a unique class of transcription factors because it contains a ligand-binding domain that, when activated, results in nuclear translocation and the transcriptional activation of genes associated with prostate cancer development. Although androgen deprivation therapies are effective initially for the treatment of prostate cancer, the disease eventually relapses and progresses to castration-resistant prostate cancer (CRPC). Nonetheless, the AR still plays a critical role because late-stage investigational agents that deplete testosterone (abiraterone) or block ligand binding (MDV3100) can still control tumor growth in patients with CRPC. These findings indicate that downmodulation of AR expression may provide a complementary strategy for treating CRPC. In this article, we describe a novel, locked, nucleic acid-based antisense oligonucleotide, designated EZN-4176. When administered as a single agent, EZN-4176 specifically downmodulated AR mRNA and protein, and this was coordinated with inhibition of the growth of both androgen-sensitive and CRPC tumors in vitro as well as in animal models. The effect was specific because no effect on growth was observed with a control antisense oligonucleotide that does not recognize AR mRNA, nor on tumors derived from the PC3, AR-negative, tumor cell line. In addition, EZN-4176 reduced AR luciferase reporter activity in a CRPC model derived from C4-2b cells that were implanted intratibially, indicating that the molecule may control prostate cancer that has metastasized to the bone. These data, together with the continued dependency of CRPC on the AR signaling pathway, justify the ongoing phase I evaluation of EZN-4176 in patients with CRPC.

Potent and sustained inhibition of HIF-1α and downstream genes by a polyethyleneglycol-SN38 conjugate, EZN-2208, results in anti-angiogenic effects.

Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFß1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208's superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208.

Marked therapeutic efficacy of a novel polyethylene glycol-SN38 conjugate, EZN-2208, in xenograft models of B-cell non-Hodgkin's lymphoma.

Examination of the clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin), the active metabolite of CPT-11, has not been possible to date due to poor solubility of SN38. Here we evaluated the activity of EZN-2208, a water-soluble polyethyleneglycol-SN38 conjugate, in pre-clinical models of Burkitt's non-Hodgkin's lymphoma (NHL) (Raji and Daudi), and follicular NHL (DoHH2). In vitro, the IC50 of EZN-2208 ranged from 3-24 nM, which was 30- to 45-fold lower than CPT-11 or 2.5- to 3.5-fold higher than SN38. In both an early-disease Raji model and an advanced-disease Daudi model, treatment with multiple doses of EZN-2208 resulted in 90% and 100% cures of animals, respectively (cure defined as no sign of tumors by gross observations at the termination of study). The activity of EZN-2208 was dramatically superior to that of CPT-11 in all three models. The excellent therapeutic efficacy of EZN-2208 in several B-cell NHL xenograft models merits its evaluation in the clinic for lymphoid malignancies.

Discovery and structure-activity relationships of a novel series of benzopyran-based K(ATP) openers for urge urinary incontinence.

A novel series of benzopyran derivatives were synthesized and evaluated as K(ATP) channel openers. Structure-activity relationships were investigated around 4-position of the benzopyran nucleus. Optimization of 4-substituent with some heterocyclic rings led to compound 13b bearing a benzo[d]isoxazol-3-one moiety as a potent and selective K(ATP) channel opener in vitro. In two anesthetized rat models of myogenic bladder overactivity, compound 13b was found to inhibit spontaneous bladder contractions.

Novel delivery of SN38 markedly inhibits tumor growth in xenografts, including a camptothecin-11-refractory model.

PURPOSE: Clinical development of SN38, the active metabolite of camptothecin-11 (CPT-11), has been hampered due to its poor solubility. We have developed a novel polymer-drug conjugate, EZN-2208, made by linking SN38 with a multiarm polyethylene glycol via a glycine linker. EXPERIMENTAL DESIGN: The in vitro cytotoxicity of EZN-2208 was tested using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. The therapeutic efficacy of EZN-2208 was evaluated in various xenografts, including an in vivo-selected CPT-11-refractory model. Tumor and blood concentration of EZN-2208, CPT-11, and SN38 was determined by high-performance liquid chromatography. RESULTS: In vitro, EZN-2208 was 10- to 245-fold more potent than CPT-11 in a panel of human tumor cell lines. In xenograft models of MX-1 breast, MiaPaCa-2 pancreatic, or HT-29 colon carcinoma, treatment with either a single dose or multiple injections of EZN-2208 was more efficacious (and in some cases produced tumor eradication for >16 weeks) compared with CPT-11 at their respective maximum tolerated doses or corresponding dose levels (P < 0.01). Most interestingly, EZN-2208 showed marked antitumor activity in animals that developed resistance to an 8-day course of CPT-11 treatment, as well as outperformed CPT-11 as second-round therapy in mice initially sensitive to CPT-11. EZN-2208 had prolonged circulation in the blood compared with CPT-11, resulting in high tumor exposure. This resulted in higher and longer-lasting tumor exposure of free SN38 in mice given EZN-2208 compared with those given CPT-11. CONCLUSIONS: Preclinical data suggest that EZN-2208 may be a promising anticancer agent in a wide variety of clinical settings, including tumors refractory to CPT-11 treatment.

Effects of protein kinase C activation on cardiac repolarization and arrhythmogenesis in Langendorff-perfused rabbit hearts.

AIMS: Cardiac arrhythmias are still a major cause of mortality in western countries. Currently available antiarrhythmic drugs are limited by a low efficacy and proarrhythmic effects. The role of the protein kinase C (PKC) signalling pathway in arrhythmogenesis is still unclear. The goal of the present study was to test the effects of PKC stimulation on whole heart electrophysiology and its pro-/antiarrhythmic activity. METHODS AND RESULTS: Left ventricular (LV) action potential duration (APD 90%) was determined in 27 Langendorff-perfused rabbit hearts, using Tyrode solution plus the PKC agonist phorbol-12-myristate-13-acetate (PMA; 100 nM) alone (nine rabbits), Verapamil alone (n = 6), or PMA in combination with Verapamil (0.25 mg/L, six rabbits), or bisindolylmaleimide (0.5 microM, n = 6). Intermittent programmed extra-stimulation was performed to induce ventricular arrhythmias. Administration of PMA alone led to a significant shortening of repolarization (APD 90%, 157 +/- 8 vs. 128 +/- 5 ms, P<0.05). Non-sustained ventricular fibrillation (VF) could be induced in seven out of nine animals. After perfusion of Verapamil (156 +/- 6 vs. 169 +/- 4 ms, P>0.05) or bisindolylmaleimide, a selective inhibitor of PKC (136 +/- 4 vs. 146 +/- 4 ms, P>0.05), PMA-induced shortening of repolarization could be inhibited, and induction of VF failed. Verapamil alone did not affect APD and VF could not be induced. CONCLUSIONS: Activation of PKC facilitates induction of VF, which is most likely due to a shortening of repolarization and a prominent calcium influx. These findings demonstrate involvement of the PKC-signalling pathway in arrhythmogenesis.

Role of KATP channels in repetitive induction of ventricular fibrillation.

AIM: Patients with sustained ventricular tachyarrhythmias are at high risk for sudden cardiac death. The mechanisms leading to multiple temporally related episodes of ventricular fibrillation (VF) are not yet fully elucidated, and treatment options are limited. We investigated whether K(ATP)-channels could be involved in triggering VF. METHODS: We determined postarrhythmic changes of monophasic action potentials (MAP) after repetitive induction of VF in 32 Langendorff-perfused rabbit hearts. RESULTS: Postarrhythmic action potential duration (APD) was significantly shorter compared with baseline (100 +/- 12 ms vs. 140 +/- 8 ms, P < 0.05). With increasing numbers of VF and shortening of recovery intervals between VF episodes (2 min) inducibility of VF increased, and abbreviation of APD became more prominent (90 +/- 5 ms vs. 130 +/- 4 ms, P < 0.05). Pre-treatment with the selective K(ATP) blocking agent HMR 1883 led to a significant increase of postarrhythmic APDs compared with control hearts (100 +/- 12 ms vs. 118 +/- 3 ms, P = 0.0013). Moreover, HMR 1883 significantly reduced inducibility of VF and increased the rate of successful defibrillation. CONCLUSIONS: Repetitive episodes of VF result in postarrhythmic abbreviation of APDs, a phenomenon thought to be of potential relevance for incessant tachyarrhythmias in patients. Prevention of postarrhythmic MAP-shortening by HMR 1883 might be useful in suppressing VF.

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.

Pyrroloquinolone PDE5 inhibitors with improved pharmaceutical profiles for clinical studies on erectile dysfunction.

We previously reported a series of potent and selective pyrimidinyl pyrroloquinolone PDE5 inhibitors such as 2a for potential use in male erectile dysfunction (MED) (Sui, Z.; Guan, J.; Macielag, M. J.; Jiang, W.; Zhang, S.; Qiu, Y.; Kraft, P., Bhattacharjee, S.; John, T. M.; Craig, E.; Haynes-Johnson, D.; Clancy, J. J. Med. Chem. 2002, 45, 4094-4096). Unfortunately, the low aqueous solubility and poor oral bioavailability rendered them undesirable development candidates. To address this issue, we designed a series of analogues using two approaches: increasing the overall basicity and reducing molecular weight of the lead. Through earlier SAR studies, we discovered that the PDE5 potency of the pyrroloquinolones is insensitive to substitution on the pyrrole nitrogen. Basic functional groups such as pyridines and benzimidazoles were appended via the aromatic ring connected to the pyrrole nitrogen. Several truncated analogues were also designed and synthesized to improve oral absorption. These modifications allowed us to identify analogues with good oral bioavailability in rats, dogs, and monkeys while the high potency against PDE5 and desirable selectivity versus other PDE isozymes were maintained. Compounds R-11e and R-11l were selected as development candidates for MED and other indications.

The new selective I(Ks)-blocking agent HMR 1556 restores sinus rhythm and prevents heart failure in pigs with persistent atrial fibrillation.

BACKGROUND: Antiarrhythmic drugs for treatment of atrial fibrillation in patients with heart failure are limited by proarrhythmia and low efficacy. Experimental studies indicate that the pure I(Ks) blocking agents chromanol 293b and HMR 1556 prolong repolarization more markedly at fast than at slow heart rates and during beta-adrenergic stimulation. These properties may overcome some of the above quoted limitations. METHODS AND RESULTS: Ten domestic swine underwent pacemaker implantation (PM) and atrial burst pacing to induce persistent AF. Four days after onset of persistent AF, pigs were randomized to HMR 1556 (30 mg/kg, p.o., 10 days) or placebo. All animals receiving HMR 1556 converted to SR (5.2 +/- 1.9 days), whereas placebo pigs remained in AF. Pigs treated with placebo developed high ventricular rates (297 +/- 5 bpm) and severe heart failure, whereas pigs treated with HMR 1556 remained hemodynamically stable. Left ventricular ejection fraction on the day of euthanization was significantly lower in the placebo compared to the HMR 1556 group (30 +/- 4% vs. 69 +/- 5%, p < 0.005). Similar results were seen with epinephrine levels (placebo 1563 +/- 193 pmol/l vs. HMR 613 +/-196 pmol/l, p < 0.05). Right atrial monophasic action potentials were significantly longer in the HMR 1556 compared to the placebo group (230 +/- 7 ms vs. 174 +/- 13 ms, p < 0.05). CONCLUSIONS: The new I(Ks) blocker HMR 1556 efficiently and safely restores SR and prevents CHF in a model of persistent AF. Restoration of SR is most likely linked to a marked prolongation of atrial repolarization even at high heart rates.

Altered activities of cyclic nucleotide phosphodiesterases and soluble guanylyl cyclase in cultured RFL-6 cells.

We utilized rat fetal lung fibroblasts (RFL-6) to evaluate our PDE5 inhibitors at cellular level and observed a decrease in cGMP accumulation induced by sodium nitroprusside (SNP) and PDE5 inhibitors with passage. To further investigate this observation, we examined cGMP synthesis via soluble guanylyl cyclase (sGC) and degradation via phosphodiesterases (PDEs) at different passages. At passage (p)4, p9, p14, major cGMP and cAMP degradation activities were contributed by PDE5 and PDE4, respectively. The PDE5 activity decreased 50% from p4 to p14, while PDE4 activity doubled. The cGMP accumulation was evaluated in the presence of sodium nitroprusside (SNP) and/or PDE inhibitors in p4 and p14 cells. SNP together with sildenafil, a PDE5 inhibitor, induced dose-dependent increase in cGMP levels in cells at p4, but showed little effect on cells at p14. The possible down regulation of sGC at mRNA level was explored using real-time RT-PCR. The result showed the mRNA level of the alpha1 subunit of sGC decreased about 98% by p9, while the change on beta1 mRNA was minimal. Consistently, sGC activities in cell lysate decreased by 94% at p9. Forskolin stimulated a dramatic increase in cAMP levels in cells at all passages examined. Our results show that sGC activity decreased significantly and rapidly with passage due to a down regulation of the alpha1 subunit mRNA, yet the adenylyl cyclase activity was not compromised. This study further emphasized the importance of considering passage number when using cell culture as a model system to study NO/cGMP pathway.

Synthesis and SAR of tetracyclic pyrroloquinolones as phosphodiesterase 5 inhibitors.

The synthesis of the fused tetracyclic pyrroloquinolones 9a-i in four steps is described. The PDE5 inhibitory activities of these compounds, their selectivities against PDE1, PDE2, PDE3, PDE4 and PDE6, the preclinical pharmacokinetic assessments and the in vivo efficacy in increasing intracavernosal pressure are presented and discussed.

Pro- and antiarrhythmic effects of fast cardiac pacing in a canine model of acquired long QT syndrome.

Increasing the heart rate is one option for suppressing bradycardia-dependent polymorphic ventricular tachycardias (PVTs). The mechanisms underlying preventive pacing in acquired forms of the long QT syndrome (LQTs) are still not fully understood. Using two needle electrodes, local effective refractory periods (ERPs) were determined in the left (LV) and right ventricle (RV) in 20 dogs with acute AV node ablation before continuous pacing, during a 20-min period of continuous fast pacing (Cl 300 ms, fastpac) and during a 35-min recovery period with slow (Cl 500 ms) pacing. This protocol was applied to control dogs (5 dogs) and dogs with pretreatment of the IKs blocking agent chromanol 293b (5 dogs, LQTs1), the IKr-blocking agent dofetilide (5 dogs, LQTs2) or a combination thereof (5 dogs). Fastpac resulted in a significant abbreviation of ERPs in control dogs and dogs receiving dofetilide or chromanol 293b. During recovery, shortening of ERPs persisted in the control group, but diminished in dogs with acquired LQTs. In dogs with LQTs2 fastpac could not suppress inhomogeneity of refractoriness during recovery. With pretreatment of dofetilide and chromanol 293b in combination, MAP duration during fastpac significantly increased (first beat: 256+/-6 ms vs. sixth beat: 278+/-9 ms, p<0.05) and fastpac-induced PVTs were evident. ERP shortening and reduced inhomogeneity of refractoriness might be one antiarrhythmic action of fastpac in dogs with acute AV-block. However, in the acquired LQTs1 and 2 beneficial effects of fastpac diminished and in a combination thereof fastpac-induced PVTs are likely.