A single preoperative blood test predicts postoperative sepsis and pneumonia after coronary bypass or open aneurysm surgery.

BACKGROUND: Major surgery comes with a high risk for postoperative inflammatory complications. Preoperative risk scores predict mortality risk but fail to identify patients at risk for complications following cardiovascular surgery. We therefore assessed the value of preoperative red cell distribution width (RDW) as a predictor for pneumonia and sepsis after cardiovascular surgery and studied the relation of RDW with hematopoietic tissue activity. METHODS: RDW is an easily accessible, yet seldomly used parameter from routine haematology measurements. RDW was extracted from the Utrecht Patient Orientated Database (UPOD) for preoperative measurements in patients undergoing open abdominal aortic anuerysm repair (AAA)(N = 136) or coronary artery bypass grafting (CABG)(N = 2193). The cohorts were stratified in tertiles to assess effects over the different groups. Generalized Linear Models were used to determine associations between RDW and postoperative inflammatory complications. Hematopoietic tissue activity was scored using fluor-18-(18F)-deoxyglucose positron emission tomography and associated with RDW using linear regression models. RESULTS: In total, 43(31.6%) and 73 patients (3.3%) suffered from inflammatory complications after AAA-repair or CABG, respectively; the majority being pneumonia in both cohorts. Postoperative inflammatory outcome incidence increased from 19.6% in the lowest to 48.9% in the highest RDW tertile with a corresponding risk ratio (RR) of 2.35 ([95%CI:1.08-5.14] P = 0.032) in AAA patients. In the CABG cohort, the incidence of postoperative inflammatory outcomes increased from 1.8% to 5.3% with an adjusted RR of 1.95 ([95%CI:1.02-3.75] P = 0.044) for the highest RDW tertile compared with the lowest RDW tertile. FDG-PET scans showed associations of RDW with tissue activity in the spleen (B = 0.517 [P = 0.001]) and the lumbar bone marrow (B = 0.480 [P = 0.004]). CONCLUSION: Elevated RDW associates with increased risk for postoperative inflammatory complications and hematopoietic tissue activity. RDW likely reflects chronic low-grade inflammation and should be considered to identify patients at risk for postoperative inflammatory complications following cardiovascular surgery.

The Impact of Diabetes and Time on the Atherosclerotic Plaque and Cardiovascular Outcome in Patients Undergoing Iliofemoral Endarterectomy.

OBJECTIVE: The incidence of diabetes is rapidly increasing and diabetes is associated with an increased risk of peripheral artery disease. Recent studies have shown a time dependent decline in vulnerable plaque features and secondary cardiovascular events in iliofemoral endarterectomy (IFE) patients. IFE patients with diabetes have a high risk of cardiovascular events. It is not known, however, whether vulnerable plaque features and cardiovascular events reduce over time in IFE patients with diabetes. METHODS: Between 2003 and 2014, 691 atherosclerotic plaques were obtained by IFE, from 212 patients with and 479 patients without diabetes. Plaques were immunohistochemically stained and analysed for the presence of intraplaque haemorrhage, lipid core, calcification, collagen, smooth muscle cells, and macrophages. Patients were stratified according to their diabetic status and year of inclusion. All patients had a follow up of three years in which cardiovascular adverse events were recorded. RESULTS: A time dependent decrease was observed in intraplaque haemorrhage, plaque lipid core, and percentage of macrophages in IFE patients with diabetes. After multivariable correction for changes in risk factors over time, intraplaque haemorrhage (64.2% [2002-2005] vs. 39.6% [2012-2014], p = .01) became significantly less prevalent. Interestingly, the percentage of severely calcified plaques remained high over time. The number of secondary events decreased over time in patients without diabetes (HR 1.80, 95% CI 1.15-2.81 (p = .010) for 2002-2005 vs. 2012-2014), but remained high and unchanged in patients with diabetes. CONCLUSION: In patients with diabetes undergoing IFE, a time dependent stabilisation of atherosclerotic plaque features was found in line with previous observations in patients with severe atherosclerosis. The presence of severely calcified lesions remained high and unchanged. The secondary event rate remained high in patients with diabetes in contrast to a significant decrease in patients without diabetes. These findings stress the need for improvement of care in IFE patients with diabetes.

Relationship of MMP-14 and TIMP-3 expression with macrophage activation and human atherosclerotic plaque vulnerability.

Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14(hi) TIMP-3(lo) rabbit foam cells are more invasive and more prone to apoptosis than MMP-14(lo) TIMP-3(hi) cells. We investigated the implications of these findings for human atherosclerosis. In vitro generated macrophages and foam-cell macrophages, together with atherosclerotic plaques characterised as unstable or stable, were examined for expression of MMP-14, TIMP-3, and inflammatory markers. Proinflammatory stimuli increased MMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages. However, conversion to foam-cells with oxidized LDL increased MMP-14 and decreased TIMP-3 protein, independently of inflammatory mediators and partly through posttranscriptional mechanisms. Within atherosclerotic plaques, MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage markers, whereas TIMP-3 was present in less foamy macrophages and colocalised with CD206. MMP-14 positive macrophages were more abundant whereas TIMP-3 positive macrophages were less abundant in plaques histologically designated as rupture prone. We conclude that foam-cells characterised by high MMP-14 and low TIMP-3 expression are prevalent in rupture-prone atherosclerotic plaques, independent of pro- or anti-inflammatory activation. Therefore reducing MMP-14 activity and increasing that of TIMP-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction.

Endovascular Treatment of Aortic Stump Rupture After Extra-anatomic Aortoduodenal Fistula Repair is not a Definitive Treatment: A Case Report and Literature Review.

Introduction: Endovascular treatment of an aortic stump rupture is technically feasible. Whether this is a definitive treatment or a bridge to further surgery is unknown. Report: Previously a Case of an aortic stump rupture following extra-anatomic repair of a recurrent aortoduodenal fistula (ADF), which was successfully treated endovascularly by placement of an Amplatzer® Vascular Plug was described. The patient survived this acute procedure, but four years later was admitted with fever and back pain. Imaging revealed progressive enlargement of the aortic stump. A re-exploration was performed with removal of the infected aortic stump including the Amplatzer plug. A new aortic stump was created together with resection of an adherent part of the duodenum. The patient was discharged after five months and was able to survive for two more years without any recurring vascular complications. Discussion: This Case demonstrates that after four years, endovascular treatment was not a definitive treatment for aortic stump rupture. Endovascular treatment should be followed by definitive treatment when the patient is fit for surgery, especially in cases of ADF. If the patient is unfit for surgery, conservative treatment with culture based antibiotics is a reasonable alternative. Positive obstinacy lengthened the survival of this patient with eight years of reasonably good quality life.

Capnocytophaga canimorsus Mycotic Aortic Aneurysm After a Dog Bite.

Introduction: Mycotic aortic aneurysm is defined as dilatation of the aortic wall due to infection caused by a variety of microorganisms and is associated with high mortality rates. This case report describes a patient with a rapid growing mycotic infrarenal aneurysm caused by Capnocytophaga canimorsus following a dog bite. Report: A 61 year old male professional dog handler presented with a history of progressive abdominal pain and constitutional symptoms. He had been bitten by a Pit Bull Terrier dog that was attacking a young girl three weeks prior to the onset of complaints. Investigations revealed a mycotic infrarenal aortic aneurysm that grew 0.5 cm in only three days. Open surgical repair consisting of an infrarenal aorto-aortic bypass with a 21 mm × 15 cm bovine bioprosthesis was performed successfully. All cultures and biopsies were negative and the subsequent 16S-23S rRNA intergenic spacer region based polymerase chain reaction (IS-pro) technique revealed C. canimorsus, a Gram negative bacterial pathogen that lives as a commensal in the gingival flora of dogs and cats that can cause a variety of severe infections, as the causative agent. Identification made it possible to treat the patient with eight weeks of intravenous followed by four weeks of oral antibiotics. At the last follow up over a year after surgery, the patient was symptom free, without infection and on ultrasound examination there were no signs of complications or aneurysm formation. Discussion: This case highlights C. canimorsus as a rare cause of a rapid growing mycotic aortic aneurysm following a dog bite. 16S-23S rRNA profiling (IS-pro) led to the identification of the bacterial pathogen. The use of biological grafts should be considered in the management of mycotic aortic aneurysms.

Toll-like receptor 2 and 4 stimulation elicits an enhanced inflammatory response in human obese patients with atherosclerosis.

The innate immune response elicited by activation of TLRs (Toll-like receptors) plays an important role in the pathogenesis of atherosclerosis. We hypothesized that cardiovascular risk factors are associated with the activation status of the innate immune system. We therefore assessed the responsiveness of TLRs on circulating cells in two groups of patients with established atherosclerosis and related this to the presence of cardiovascular risk factors. TNF (tumour necrosis factor)-α release induced by TLR2 and TLR4 activation was measured in patients with established coronary [PCI (percutaneous coronary intervention) study, n=78] or carotid artery disease [CEA (carotid endarterectomy) study, n=104], by stimulating whole blood samples with lipopolysaccharide (TLR4 ligand) and Pam3CSK4 [tripalmitoylcysteinylseryl-(lysyl)4; TLR2 ligand]. As an early activation marker, CD11b expression was measured by flow cytometry on CD14+ cells. Obesity was the 'only' risk factor that correlated with the TLR response. In both studies, obese patients had significantly higher TNF-α levels after stimulation of TLR2 compared with non-obese patients [16.9 (7.7-49.4) compared with 7.5 (1.5-19.2) pg/ml (P=0.008) in coronary artery disease and 14.6 (8.1-28.4) compared with 9.5 (6.1-15.7) pg/ml (P=0.015) in carotid artery disease; values are medians (interquartile range)]. Similar results were obtained following TLR4 stimulation. The enhanced inflammatory state in obese patients was also confirmed by a significant increased expression of the activation marker CD11b on circulating monocytes. In conclusion, obesity is associated with an enhanced TLR response in patients suffering from established atherosclerotic disease.

Trait anxiety predicts outcome 6 weeks after cholecystectomy. A prospective follow-up study.

BACKGROUND: A substantial group of patients with gallstone disease experience negative outcome after surgical removal of the gallbladder (cholecystectomy). Early identification of these patients is important. PURPOSE: The aim of the study is to identify predictors (clinical symptoms and trait anxiety) of negative symptomatic outcomes at 6 weeks after cholecystectomy. METHODS: Consecutive patients (n = 133), 18-65 years, with symptomatic gallstone disease, completed symptom checklists and the state-trait anxiety inventory preoperatively and at 6 weeks after cholecystectomy. RESULTS: High trait anxiety was the only predictor of persistence of biliary symptoms at 6 weeks after cholecystectomy (OR = 6.88). CONCLUSION: In addition to clinical symptoms, high trait anxiety is a predictor of negative symptomatic outcome at 6 weeks after cholecystectomy. Trait anxiety should be evaluated to aim at a patient-tailored approach in gallstone disease.

Trait anxiety predicts unsuccessful surgery in gallstone disease.

OBJECTIVE: To identify predictors of negative symptomatic outcomes at 6 months after cholecystectomy, surgical removal of the gallbladder, which is the preferred treatment for gallstone disease. After cholecystectomy, a substantial number of patients report persistence of symptoms. METHODS: In this prospective follow-up study, consecutive patients (n = 172) diagnosed with symptomatic gallstone disease and indicated for elective cholecystectomy were investigated. Preoperatively and at 6 months, patients completed self-report symptom checklists. The Spielberger State-Trait Anxiety Inventory scale was completed preoperatively and patients with a score of > or = P 80 were considered having High Trait Anxiety (HTA). Multivariate regression analyses were used to investigate independent predictors of persisting symptoms. RESULTS: Six months after cholecystectomy, patients with HTA were more likely to report persisting biliary symptoms than patients without HTA (NHTA) (45.5% versus 14.3%; chi(2) = 8.78, p = .002). HTA was identified as an independent predictor of persisting biliary symptoms at 6 months (odds ratio [OR], 3.08, p = .047; 95% confidence interval [CI], 1.02-9.34), in addition to the report of nonspecific symptoms (OR, 6.16, p = .024; 95% CI, 1.27-29.82), and the use of psychotropic medication (OR, 4.76, p = .023; 95% CI, 1.24-18.34). CONCLUSION: Patients with HTA have a three times higher risk at persisting biliary symptoms at 6 months after cholecystectomy than NHTA patients. Both clinical factors and the patient's personality should be considered in clinical decision making and risk estimation in elective cholecystectomy.

Pharmacologic/pharmacokinetic evaluation of emesis induced by analogs of RSU 1069 and its control by antiemetic agents.

RB 6145, the ring-opened analog of RSU 1069, and PD 130908, the desoxy ring-opened analog of RSU 1069, were compared to RSU 1069 for their emetic potential in dogs. When RB 6145 and PD 130908 were administered intravenously at doses ranging from 20% to 50% of the mouse equivalent maximum tolerated dose (MTD), both analogs were less emetic than RSU 1069 on a molar basis. Furthermore, the 5HT3 antagonist ondansetron prevented emesis at doses as high as 75% of the MTD. The reactivity, and hence the emetic liability of these compounds, is thought to be mediated by formation of the corresponding aziridine intermediate. In mouse plasma, both analogs rapidly converted to two products, the reactive aziridine and a stable oxiazolidinone species formed upon reaction with bicarbonate in the blood. A positive correlation exists between the amounts of aziridine formed by these analogs and their emetic potential.

Tyrosine kinase inhibitors. 13. Structure-activity relationships for soluble 7-substituted 4-[(3-bromophenyl)amino]pyrido[4,3-d]pyrimidines designed as inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor.

The general class of 4-(phenylamino)quinazolines are potent (some members with IC50 values << 1 nM) and selective inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), via competitive binding at the ATP site of the enzyme, but many of the early analogues had poor aqueous solubility (<< 1 mM). A series of 7-substituted 4-[(3-bromophenyl)-amino]pyrido[4,3-d]pyrimidines, together with selected (3-methylphenyl)amino analogues, were prepared by reaction of the analogous 7-fluoro derivatives with appropriate amine nucleophiles in 2-BuOH or aqueous 1-PrOH. All of the compounds were evaluated for their ability to inhibit the tyrosine-phosphorylating action of EGF-stimulated full-length EGFR enzyme. Selected analogues were also evaluated for their inhibition of autophosphorylation of the EGF receptor in A431 human epidermoid carcinoma cells in culture and against A431 tumor xenografts in mice. Analogues bearing a wide variety of polyol, cationic, and anionic solubilizing substituents retained activity, but the most effective in terms of both increased aqueous solubility (> 40 mM) and retention of overall inhibitory activity (IC50's of 0.5-10 nM against isolated enzyme and 8-40 nM for inhibition of EGFR autophosphorylation in A431 cells) were weakly basic amine derivatives. These results are broadly consistent with a proposed model for the binding of these compounds to EGFR, in which the 6- and 7-positions of the pyridopyrimidine ring are in a largely hydrophobic binding region of considerable steric freedom, at the entrance of the adenine binding cleft. The most active cationic analogues have a weakly basic side chain where the amine moiety is three or more carbon atoms away from the nucleus. Two of the compounds (bearing weakly basic morpholinopropyl and strongly basic (dimethylamino)butyl solubilizing groups) produced in vivo tumor growth delays of 13-21 days against advanced stage A431 epidermoid xenografts in nude mice, when administered i.p. twice per day on days 7-21 posttumor implant. Treated tumors did not increase in size during therapy and resumed growth at the termination of therapy, indicating an apparent cytostatic effect for these compounds under these treatment conditions. The data suggest that continuous long-term therapy with these compounds may result in substantial tumor growth inhibition.

Tyrosine kinase inhibitors. 14. Structure-activity relationships for methylamino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors.

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.

Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides bearing additional solubilizing functions.

4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepared by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino-7-alkylamines were prepared from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogues with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC(50[app]) values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2-d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6-acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogues showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the observed lower cellular potency of the pyrido[3,2-d]pyrimidine-6-acrylamides. Selected compounds showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogues in terms of aqueous solubility, potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clinical evaluation.

Tyrosine kinase inhibitors. 18. 6-Substituted 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as soluble, irreversible inhibitors of the epidermal growth factor receptor.

4-Anilinoquinazoline- and 4-anilinopyrido[3,4-d]pyrimidine-6-acrylamides are potent pan-erbB tyrosine kinase inactivators, and one example (CI-1033) is in clinical trial. A series of analogues with a variety of Michael acceptor units at the 6-position were prepared to define the structural requirements for irreversible inhibition. A particular goal was to determine whether additional functions to increase solubility could be appended to the Michael acceptor. Substituted acrylamides were prepared by direct acylation of the corresponding 6-amines with the requisite acid or acid chloride. Vinylsulfonamide derivatives were obtained by acylation of the amines with chloroethylsulfonyl chloride followed by base-promoted elimination. Vinylsulfone and vinylsulfine derivatives were prepared by oxidation and base elimination of a hydroxyethylthio intermediate. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Substitution at the nitrogen of the acrylamide was tolerated only with a methyl group; larger substituents were dystherapeutic, and no substitution at all was tolerated at the acrylamide alpha-carbon. In contrast, while electron-donating groups at the acrylamide beta-carbon were not useful, even quite large electron-withdrawing groups (which increase its electrophilicity) were tolerated. A series of derivatives with solubility-enhancing substituents linked to the acrylamide beta-carbon via amides were potent irreversible inhibitors of isolated EGFR (IC50s = 0.4-1.1 nM), with weakly basic morpholine and imidazole derivatives being the best. Vinylsulfonamides were also potent and irreversible inhibitors, but vinylsulfones and vinylsulfines were reversible and only poorly active. Two compounds were evaluated against A431, H125, and MCF-7 xenografts in nude mice but were inferior in these assays to the clinical trial compound CI-1033.

Soluble 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas. Structure-activity relationships against selected tyrosine kinases and exploration of in vitro and in vivo anticancer activity.

In continuing our search for medicinal agents to treat proliferative diseases, we have discovered 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 1, several series of analogues were made that examined the C-6 aryl substituent, a variety of water solublizing substitutents at the C-2 position, and urea or other acyl functionality at the N-7 position. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr;) and nonreceptor (c-Src) classes. Several of the most potent compounds displayed submicromolar inhibition of PDGF-mediated receptor autophosphorylation in rat aortic vascular smooth muscle cells and low micromolar inhibition of cellular growth in five human tumor cell lines. One of the more thoroughly evaluated members, 32, with IC50 values of 0.21 microM (PDGFr), 0.049 microM (bFGFr), and 0.018 microM (c-Src), was evaluated in in vivo studies against a panel of five human tumor xenografts, with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs. Compound 32 produced a tumor growth delay of 14 days against the Colo-205 colon xenograft model.

Tyrosine kinase inhibitors. 6. Structure-activity relationships among N- and 3-substituted 2,2'-diselenobis(1H-indoles) for inhibition of protein tyrosine kinases and comparative in vitro and in vivo studies against selected sulfur congeners.

A small series of 2,2'-diselenobis(1H-indoles) was synthesized as redox-modified congeners of our earlier reported 2,2'-dithiobis(1H-indole) series. Utilizing chemistry similar to that developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indolecarboxylic acid precursors bearing various polar functionality at the C-3 position and small alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic source of diselenium, and a much improved process to a 2,2'-dithiobis(1H-indole) congener was developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine kinases, compounds in this series displayed broad inhibitory activity with IC50 = 0.9 to > 100 microM vs EGFr, 3.4 to > 50 microM vs PDGFr, and 0.4-6.7 microM vs v-src. In general, compounds derived from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-indolecarboxylic acids greater potency against PDGFr and v-src. Enzyme kinetics studies showed that both classes of compounds display primarily noncompetitive inhibition with respect to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium and disulfur series with the reversal of enzyme inhibition occurring less readily within the diselenium series. In whole cell studies, compounds of this class were growth inhibitory against Swiss 3T3 mouse fibroblasts with IC50 values from 0.5 to 19.5 microM, and the observed SAR was different from that of the 2,2'-dithiobis(1H-indoles). A comparative study in the same cell line on the effects of the 2,2'-diselenobis(1H-indole) derived from (R)-tryptophan vs its disulfur congener on growth factor mediated tyrosine phosphorylation showed that this compound significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with complete suppression at 25 and 5 microM, respectively. Tyrosine phosphorylation of an 85 kDa protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at submicromolar concentrations. The disulfur congener exhibited a qualitatively similar pattern; however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses (1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was effective against the C6 glioma when administered intraperitoneally on a d1-9 schedule. Studies were also carried out against the A431 epidermoid xenograft to evaluate the same pair of compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The maximum dose that could be administered daily was limited by compound solubility. Neither compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-day growth delay without appreciable treatment related weight loss. The in vitro and in vivo findings offer a mechanistic rationale as to why the 2,2'-diselenobis(1H-indoles) are more potent inhibitors than their disulfur congeners.

Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

3-(3,5-Dimethoxyphenyl)-1,6-naphthyridine-2,7-diamines and related 2-urea derivatives are potent and selective inhibitors of the FGF receptor-1 tyrosine kinase.

A series of 3-aryl-1,6-naphthyridine-2,7-diamines and related 2-ureas were prepared and evaluated as inhibitors of the FGF receptor-1 tyrosine kinase. Condensation of 4,6-diaminonicotinaldehyde and substituted phenylacetonitriles gave intermediate naphthyridine-2,7-diamines, and direct reaction of the monoanion of these (NaH/DMF) with alkyl or aryl isocyanates selectively gave the 2-ureas in varying yields (23-93%). For the preparation of more soluble 7-alkylamino-2-ureas, a number of protecting groups for the 2-amine were evaluated (phthaloyl, 4-methoxybenzyl) following selective blocking of the 7-amine (trityl), but these were not superior to the (required) 2-tert-Bu-urea group itself. Direct alkylation of the anion of the (unprotected) 7-amino group with excess 4-(3-chloropropyl)morpholine in DMF gave low (10%) yields of the desired product, but alkylation of the 7-acetamido anion, followed by mild alkaline hydrolysis, raised this to 64%. 3-Phenyl analogues were nonspecific inhibitors of isolated c-Src, FGFR, and PDGFR tyrosine kinases, whereas 3-(2,6-dichlorophenyl) analogues were most effective against c-Src and FGFR, and 3-(3,5-dimethoxyphenyl) derivatives showed high selectivity for FGFR alone. A water-soluble (7-morpholinylpropylamino) analogue retained high FGFR potency (IC(50) 31 nM) and selectivity. Pairwise comparison of the 1, 6-naphthyridines and the corresponding known pyrido[2,3-d]pyrimidine analogues showed little differences in potency or patterns of selectivity, suggesting that the 1-aza atom of the latter is not important for activity. A 7-acetamide derivative inhibited the growth of FGFR-expressing tumor cell lines and was particularly potent against HUVECs (IC(50) 4 nM). This compound was also a very potent inhibitor of HUVEC microcapillary formation (IC(50) 0.01 nM) and Matrigel invasion (IC(50) 7 nM) and showed significant in vivo antitumor effects in a highly vascularized mammary adenocarcinoma 16/c model at nontoxic doses. The compounds are worthy of further evaluation as antiangiogenesis agents.

2-Substituted aminopyrido[2,3-d]pyrimidin-7(8H)-ones. structure-activity relationships against selected tyrosine kinases and in vitro and in vivo anticancer activity.

While engaged in therapeutic intervention against a number of proliferative diseases, we have discovered the 2-aminopyrido[2, 3-d]pyrimidin-7(8H)-ones as a novel class of potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 2, a series of analogues bearing variable substituents at the C-2 position and methyl or ethyl at N-8 was made. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr; epidermal growth factor, EGFr) and nonreceptor (c-Src) classes. One of the more thoroughly evaluated members was 63 with IC50 values of 0.079 microM (PDGFr), 0.043 microM (bFGFr), 0.044 microM (EGFr), and 0.009 microM (c-Src). In cellular studies, 63 inhibited PDGF-mediated receptor autophosphorylation in a number of cell lines at IC50 values of 0.026-0.002 microM and proliferation of two PDGF-dependent lines at 0.3 microM. It also caused inhibition of soft agar colony formation in three cell lines that overexpress the c-Src TK, with IC50 values of 0.33-1.8 microM. In in vivo studies against a panel of seven xenograft tumor models with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs, compound 63 produced a tumor growth delay of 10.6 days against the relatively refractory SK-OV-3 ovarian xenograft and also displayed activity against the HT-29 tumor. In rat oral bioavailability studies, compound 63 plasma concentrations declined in a biexponential manner, and systemic plasma clearance was high relative to liver blood flow. Finally, in rat metabolism studies, HPLC chromatography identified two metabolites of 63, which were proved by mass spectrometry and synthesis to be the primary amine (58) and N-oxide (66). Because of the excellent potency of 63 against selected TKs, in vitro and in vivo studies are underway for this compound in additional tumor models dependent upon PDGFr, FGFr, and c-Src to assess its potential for advancement to clinical trials.

Anticancer efficacy of the irreversible EGFr tyrosine kinase inhibitor PD 0169414 against human tumor xenografts.

PURPOSE: The involvement of the EGF receptor (EGFr) family of receptors in cancers suggests that a selective inhibitor of the tyrosine kinase activity of the EGFr family could have a therapeutic effect. PD 0169414, an anilinoquinazoline, is a potent irreversible inhibitor of the EGFr family tyrosine kinase activity with IC(50) values of 0.42 nM against the isolated EGF receptor, and 4.7 nM and 22 nM against EGF- and heregulin-mediated receptor phosphorylation in A431 and MDA-MB-453 cells, respectively. METHODS AND RESULTS: Oral administration of 260 mg/kg per day PD 0169414 for 15 days to animals bearing advanced-stage A431 epidermoid carcinoma produced a 28.2-day delay in tumor growth and resulted in three complete and three partial tumor regressions in six animals. Toxicity at this dose level was limited to <6% loss of initial body weight. Doses of 160 and 100 mg/kg per day produced tumor growth delays of 29.5 and 20.9 days and two and one complete regressions in six animals, respectively. Subcutaneous, intraperitoneal, and oral routes of administration have also shown in vivo antitumor activity of PD 0169414 in a panel of human tumor xenografts. Responsive tumor lines include A431 (human epidermoid carcinoma), H125 (NSCL carcinoma), MCF-7 and UISO-BCA1 (human breast carcinoma), and SK-OV-03 (human ovarian carcinoma). The therapeutic effect ranged from delayed tumor growth (6.4 days delayed tumor growth for 14 days of treatment) to tumor regressions (32.2 days delayed tumor growth and five partial regressions in six animals) in these model systems. CONCLUSION: PD 0169414 is a specific, irreversible inhibitor of EGFr family tyrosine kinases with significant in vivo activity against a variety of relevant human tumor xenografts.

Type 2 diabetes is not associated with an altered plaque phenotype among patients undergoing carotid revascularization. A histological analysis of 1455 carotid plaques.

AIMS: Diabetes accelerates progression of atherosclerotic disease, but data on associations between diabetes and advanced atherosclerotic plaque composition are scarce. METHODS AND RESULTS: We used one of the largest biobanks, the Athero-Express study (n=1455) at carotid endarterectomy (CEA). All plaques were subjected to histological analysis to assess lipid core size, collagen, macrophages, smooth muscle cells, micro-vessel density and calcifications. In addition, within a subset of patients cytokines and chemokines were assessed. The 295 patients (20%) with type-2 diabetes showed a higher proportion of previous cardiovascular interventions and more stringent treatment for hypertension and hypercholesterolaemia compared with patients without type-2 diabetes. Surprisingly, no associations between diabetes and histological plaque characteristics were observed. In addition, no differences were observed in the expression of inflammatory chemokines, cytokines or advanced glycation end products in plaques of diabetic and non-diabetic patients. CONCLUSION: In patients suffering from significant carotid artery disease, diabetes does not appear to be associated with specific atherosclerotic plaque characteristics.