In vitro assessment of the selectivities of various beta-adrenergic blocking agents

Intrinsic sympathomimetic- and membrane activities of beta-adrenoceptor blocking agents are of little or no clinical significance. A selective blockade of cardiac receptors has important therapeutic consequences, especially in the treatment of patients with obstructive airways diseases. Profound depression of miocardial contractility can be deleterious in patients with cardiac muscle damage and the use of beta-adrenergic blockers with a quantitative selectivity towards chronotropism may become an important consideration. The effects of a number of beta-sympatholytics have been determined on isolated cardiac preparations (beta₁-adrenergic receptors) and tracheal preparations (beta₂-adrenergic receptors) of guinea-pigs. Results indicate that prindolol is the most selective blocker of the beta₁ chronotropic receptors whilst atenolol could be classified as being the most cardioselective beta-adrenergic blocker investigated. Butoxamine, on the other hand, proved to be the most beta₂-selective one.     

Beta1 and beta2 receptor mechanisms in rat jugular veins: differences between norepinephrine and isoproterenol-induced relaxation.

The rat jugular vein possesses both beta₁ and beta₂ adrenergic receptors based on the use of two beta₁ antagonists, practolol and atenolol and two beta₂ antagonists, butoxamine and N-isopropylmethoxamine. In this vessel, norepinephrine and nylidrin interact primarily with beta₁ receptors whereas isoproterenol and salbutamol interact with both beta₁ and beta₂ receptors showing a slight preference for beta₂ receptors. Isoxsuprine-induced relaxation was not blocked by either beta₁ or beta₂ antagonists. Selectivity of norepinephrine for beta₁ receptors and of isoproterenol for beta₂ receptors also occurred in circular preparations of the portal vein after alpha adrenergic blockade. However, after alpha adrenergic blockade in rat aorta, practolol and N-isopropylmethoxamine were equieffective as antagonists of relaxation to norepinephrine and isoproterenol although N-isopropylmethoxamine was somewhat more effective than practolol.     

Role of coxsackievirus B4 in the pathogenesis of acute glomerulonephritis

Coxsackievirus B₄ was isolated from the throat, nose, blood, stools and urine of a 9-year-old boy with acute glomerulonephritis and a pneumonitis. Neutralization test showed a greater than fourfold rise in the antibody titre to coxsackievirus B₄. The antistreptolysin O titre was elevated, but the complement component was within the normal range. The importance of the coxsackievirus B₄ in the pathogenesis of acute glomerulonephritis is clearly indicated; however, further investigations are needed to understand the details of the virus-kidney interaction.     

Beta2-microglobulin removal by extracorporeal renal replacement therapies

There is increasing evidence that end-stage renal disease patients with lower beta₂-microglobulin plasma levels and patients on convective renal replacement therapy are at lower mortality risk. Therefore, an enhanced beta₂-microglobulin removal by renal replacement procedures has to be regarded as a contribution to a more adequate dialysis therapy. In contrast to high-flux dialysis, low-flux hemodialysis is not qualified to eliminate substantial amounts of beta₂-microglobulin. In hemodialysis using modern high-flux dialysis membranes, a beta₂-microglobulin removal similar to that obtained in hemofiltration or hemodiafiltration can be achieved. Several of these high-flux membranes are protein-leaking, making them suitable only for hemodialysis due to a high albumin loss when used in more convective therapy procedures. On-line hemodiafiltration infusing large substitution fluid volumes represents the most efficient and innovative renal replacement therapy form. To maximize beta₂-microglobulin removal, modifications of this procedure have been proposed. These modifications ensure safer operating conditions, such as mixed hemodiafiltration, or control albumin loss at maximum purification from beta₂-microglobulin, such as mid-dilution hemodiafiltration, push/pull hemodiafiltration or programmed filtration. Whether these innovative hemodiafiltration options will become accepted in clinical routine use needs to be proven in future.     

Agonist Binding to Mammalin Beta1 and Beta2 Adrenoceptors: Modulation by Guanine Nucleotides and Magnesium

Abstract

Agonist interaction with beta₁ and beta₂ adrenoceptors in rat rabbit lung has been examined using ligand binding techniques and the results are discussed in relation to current models of beta-adrenoceptor-adenylate cyclase coupling. Agonist binding has been assessed by examining the ability of isoprenaline or salbutamol to displace the labelled antagonist ³H-dihydroalprenolol (³H-DHA) from specific receptor sites. beta₁ and beta₂ adrenoceptors, even within the same tissue, exhibited different ion and temperature requirements for guanine nucleotide modulation of agonist binding. Thus, in contrast to the situation at beta₂ sites, agonist binding to beta₁ adrenoceptors was only sensitive to GTP if incubations were performed at physiological temperatures in the presence of Mg++ ions. These findings suggest that there may be different receptor-effector coupling relationships between the two beta-adrenoceptor subtypes.     

Plasma C3 and C4 Concentrations in Management of Glomerulonephritis

As part of a larger study of serial complement profiles in glomerulonephritis plasma C3 and C4 concentrations were measured using commercially available immunodiffusion plates. A total of 303 samples were obtained from 128 patients suffering from forms of nephritis associated with hypocomplementaemia—namely, lupus nephritis, mesangiocapillary glomerulonephritis (M.C.G.N.), and acute glomerulonephritis.These simple measurements of C3 and C4 gave clinically useful information. In lupus nephritis C3 and C4 generally correlated and C4 concentrations were more often and more profoundly depressed than C3 concentrations. Neither C3 nor C4 concentrations alone correlated well with the antinuclear factor titre.In both acute glomerulonephritis and M.C.G.N. the C3 concentrations were frequently lower than 20% of normal (which was never the case in patients with lupus), while the C4 concentration was usually normal and was almost never depressed in the absence of C3 depression. This suggests activation of complement at the C3 level by the “bypass” pathway in acute nephritis as well as in M.C.G.N., though both may be operating in some patients. In acute glomerulonephritis but not in M.C.G.N. C3 concentrations returned to normal within eight to 12 weeks.The two varieties of M.C.G.N. identified by the site of the deposits in the capillary glomerular walls differed in their C3 levels. In 10 patients with intramembranous dense linear deposits the C3 was always low over very long periods of time, rising in three out of four patients only after transplantation and immunosuppression. Other patients with M.C.G.N., in contrast, often showed normal C3 concentrations. Concentrations of C4 did not differ in either group, being normal in 80% of samples from all types.     

INHIBITORY AND ANTI-INHIBITORY FACTORS OF RAT SERUM ACTIVE ON THE G1-S TRANSITION OF HEPATOCYTE CELL CYCLE

Rat hepatocytes are responsive to a serum factor inhibiting their progression through the cell cycle from the late G₁ phase to the S phase. After fractionation of normal adult rat serum by two chromatographic steps on DEAE cellulose and sephadex gel filtration, the inhibitory activity was linked to proteins having a high electronegative charge and of apparent high molecular weight. Polyacrylamide gel electrophoretic analysis of active fraction showed that the α₁ macroglobulin was its main component. Male and female baby rats were sensitive to the inhibitory factor from normal rats. Contrary to the normal adult rat serum the whole hepatectomized adult rat serum did not exhibit any inhibitory activity on the G₁-S transition. However, two components having antagonist activities: an α₁ globulin and a γ globulin, were separated by chromatographic procedures from hepatectomized rat serum.

• a. The α₁ globulin showed an inhibitory activity. It had an apparent molecular weight lower than that found in normal rats. Its activity was sex related: only male baby rats were responsive.
• b. The factor present in the γ globulin fraction was found to be antagonistic to the α₁ globulin factor. Its occurrence after hepatectomy explains the absence of inhibitory activity in the serum of hepatectomized rats.

     

Symptomless Haematuria in Childhood

The clinical, laboratory, and renal biopsy findings in 47 children with symptomless haematuria are reported. In 41 the haematuria was recurrent. Local causes were excluded by means of intravenous urography, which was normal in all but one child, who had a horseshoe kidney.Since all the patients had presented in a similar manner they were classified into four groups according to the severity of glomerular changes on renal biopsy. In group I the glomeruli were optically normal. In group 2 they showed a variable degree of mesangial thickening with absent or minimal cellular proliferation. In group 3 there was diffuse mesangial thickening and proliferation—an appearance indistinguishable from that of subsiding post-streptococcal glomerulonephritis. Compared with groups 1 and 2, more patients in this group had persistent proteinuria, as well as evidence of streptococcal infection preceding the initial haematuria. Only two patients showed severe proliferative glomerulonephritis on biopsy (group 4); both had heavy proteinuria and one repeatedly had low serum β1_c-globulin levels.     

Carbamate derivatives of colchicine show potent activity towards primary acute lymphoblastic leukemia and primary breast cancer cells—in vitro and ex vivo study

Colchicine ( COL ) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N‐carbamates of N‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti‐proliferative activity than COL (IC₅₀ = 8.6 nM) towards primary ALL cells in cell viability assays (IC₅₀ range of 1.1‐6.4 nM), and several were more potent towards primary IDCG3 (IC₅₀ range of 0.1 to 10.3 nM) or MC (IC₅₀ range of 2.3‐9.1 nM) compared to COL (IC₅₀ of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non–small‐cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.     

Steatorrhea in patients with liver disease

Intestinal function was studied in 26 patients with seven types of acute and chronic liver disease, documented by liver biopsy. Steatorrhea, defined by a stool fat higher than 6 g. per day, was present in 18 of 23 consecutive patients studied, an incidence of 78.3%. Two patients with infectious hepatitis associated with steatorrhea studied previously were added and the 20 cases were analyzed. The malabsorption found was confined to fat and fat-soluble vitamins; stool excretion varied from 6.1 to 22 g. per day in the seven groups studied. No histological abnormality was seen on jejunal biopsy, serum vitamin B₁₂, D-xylose and Schilling tests were normal, and no radiological findings associated with malabsorption were detected in the small bowel. It is concluded that steatorrhea is a common finding in a wide variety of acute and chronic liver diseases and cannot be attributed to a primary defect of the small bowel.     

Leucocyte Antigens and Disease: 1. Association of HL-A2 and Chronic Glomerulonephritis

The HL-A2 leucocyte antigen was found to be present in a significantly higher percentage of 485 patients with chronic glomerulonephritis than in 428 normal control subjects. No correlation could be shown between any of the ABO groups and chronic glomerulonephritis. There was no statistically significant difference in the frequency of HL-A2 when the controls were compared with 280 patients with a variety of other diseases. The trend toward an excess of HL-A2 positives among patients with glomerulonephritis was also seen in a study of 108 sibships, though the difference fell short of statistical significance. It was concluded that an HL-A2 positive person was about 1·5 times as likely to develop chronic glomerulonephritis as an HL-A2 negative person.     

Polypeptide marker and disease patterns found while mapping proteins in ascitis

To assess the protein composition of ascitis, 28 samples of ascitic fluid were obtained from patients admitted to Geneva University Hospital. The samples were analysed randomly and blindly by high-resolution two-dimensional polyacrylamide gel electrophoresis. The final visual evaluation was compared with the discharged summary and diagnosis. The protein pattern of ascitis was, as expected, very similar to normal or diseased plasma, with the exception of two spots which were present in ascitic fluids but not in the 200 plasma samples analyzed in parallel. After microsequencing, they proved to be β-fibrinogen fragments. Several diseases showed distinct patterns, especially acute pancreatitis. A group of intense spots with an apparent molecular mass between those of α₁-antitrypsin and β-haptoglobin were found in all ascitic fluid from pancreatitis cases (six patients). These spots had isoelectric points similar to those of α₁-antitrypsin and β-haptoglobin and microsequencing revealed that they were three different fragments of α₁-antitrypsin.     

The Fluorescent Antibody Method in the Study of Immunopathologic Conditions

Histochemical studies of immunopathologic conditions were carried out, using Coons'' fluorescent antibody technique. Experimental conditions studied were: serum sickness, generalized anaphylaxis, the Arthus reaction and experimental glomerulonephritis. Human diseases studied were those referred to as “collagen diseases”. Specific immunologic reactants were localized in the lesions of all experimental conditions studied, thus offering objective evidence of a possible immunologic pathogenesis of the lesions. In human diseases, gamma globulin was localized in the lesions of rheumatic fever, rheumatoid arthritis, systemic lupus erythematosus and amyloidosis. Although the finding of gamma globulin in human lesions might suggest that it is an antibody, such an interpretation should be made with care since the gamma globulin could be deposited on a non-immunologic basis.The tissue-localizing properties of sera from different disease states showed appreciable variability within a given disease, as well as similar localizing properties among sera of different diseases. It is suggested that these serum factors (“autoantibodies”) might result as a host response and are not primarily involved in the pathogenesis of the disease.     

Synthesis of (1,3,4-thiadiazol-2-yl)-acrylamide derivatives as potential antitumor agents against acute leukemia cells

A lead compound with the (1,3,4-thiadiazol-2-yl)-acrylamide scaffold was discovered to have significant cytotoxicity on several tumor cell lines in an in-house cell-based screening. A total of 60 derivative compounds were then synthesized and tested in a CCK-8 cell viability assay. Some of them exhibited improved cytotoxic activities. The most potent compounds had IC₅₀ values of 1–5 μM on two acute leukemia tumor cell lines, i.e. RS4;11 and HL-60. Flow cytometry analysis of several active compounds and detection of caspase activation indicated that they induced caspase-dependent apoptosis. It was also encouraging to observe that these compounds did not have obvious cytotoxicity on normal cells, i.e. IC₅₀ > 50 μM on HEK-293T cells. Although the molecular targets of this class of compound are yet to be revealed, our current results suggest that this class of compound represents a new possibility for developing drug candidates against acute leukemia.     

Characterization of Rat Cerebral Cortical Beta Adrenoceptor Subtypes Using (-)-[125I]-Iodocyanopindolol

Abstract

(-)-[¹²⁵I]-Iodocyanopindolol ((-)ICYP), used to characterize beta adrenoceptors on membrane preparations from rat cerebral cortex, was shown to have affinity for both beta adrenoceptors and serotonin receptors. Therefore, 10 µM serotonin was added to the assays to prevent (-)ICYP binding to serotonin receptors. Under these conditions, (-)ICYP binding to the cortical membrane preparation was reversible and saturable, and the association reaction was very slow. The dissociation reaction was also very slow, and revealed two affinity states corresponding to a high and a low affinity state. Scatchard analysis showed a single class of binding sites with an equilibrium dissociation constant (K_D) of 20.7 pM, and a maximal density of binding sites (B_max) of 95.1 fmol/mg membrane protein. Displacement binding analyses revealed a potency series of (-) isoproterenol greater than (-) epinephrine equal to (-) norepinephrine, suggesting a predominance of the beta₁ adrenoceptor subtype. Detailed competition ligand binding studies with the selective beta₁ adrenoceptor antagonist ICI-89406 and the selective beta₂ adrenoceptor antagonist ICI-118551, showed that about 70% of the beta adrenoceptor population in the rat cortex is of the beta₁ subtype with the remainder being of the beta₂ subtype.

We conclude that since (-)ICYP binds to both beta adrenoceptors and serotonin receptors, it is important to prevent the binding of (-)ICYP to serotonin receptors by adding a suppressing ligand like excess cold serotonin when assaying beta adrenoceptors. We have presented the first such characterization of rat cerebral cortical beta adrenoceptors with (-)ICYP in this study.     

慢性肾小球肾炎患者血清HGF、Cys-C、TAFI水平变化及其临床诊断价值

目的:分析慢性肾小球肾炎患者血清肝细胞生长因子(HGF)、胱抑素C(Cys-C)、凝血酶激活的纤溶抑制物(TAFI)水平的变化及其临床诊断价值。方法:选择我院2017年1月～2018年5月收治的71例慢性肾小球肾炎患者作为慢性肾小球肾炎组及同期于本院进行健康体检的83例作为健康对照组。检测进而比较两组血清HGF、Cys-C、TAFI水平,分析以上指标和患者肾功能的相关性及对慢性肾小球肾炎的诊断价值。结果:慢性肾小球肾炎组血清HGF、Cys-C、TAFI水平均显著高于对照组(P0.05)。慢性肾小球肾炎患者治疗后血清HGF、Cys-C、TAFI水平均显著低于治疗前(P0.05)。慢性肾小球肾炎患者血清HGF、Cys-C、TAFI水平和肾功能指标(肌酐(Scr)、尿素氮(BUN)、尿酸(UA))均呈显著正相关(P均0.05)。血清HGF水平诊断慢性肾小球肾炎的曲线下面积为0.826,敏感度和特异度分别为0.747和0.746;血清Cys-C水平诊断慢性肾小球肾炎的曲线下面积为0.821,敏感度和特异度分别为0.687和0.859;血清TAFI水平诊断慢性肾小球肾炎的曲线下面积为0.816,敏感度和特异度分别为0.855和0.647;血清HGF、Cys-C、TAFI水平联合检测诊断慢性肾小球肾炎的曲线下面积为0.951,敏感度和特异度分别为0.831和0.757。结论:慢性肾小球肾炎患者血清HGF、Cys-C及TAFI水平均明显升高,联合检测血清HGF、Cys-C及TAFI可能作为慢性肾小球肾炎诊断及预评估参考指标。     

Identification of solubilised beta1 and beta2 adrenoceptors in mammalian lung

The properties of beta-adrenoceptors in solubilised and particulate preparations of rat and rabbit lung have been assessed using the specific ligand (³H)-dihydroalprenolol ((³H)-DHA). Membranes were solubilised using the detergent digitonin and the specific binding of (³H)-DHA assayed using a charcoal-centrifugation technique to separate free and bound ligand. The equilibrium dissociation constant (K_D) of specific (³H)-DHA binding was very similar in particulate and soluble preparations of rat and rabbit lung. Moreover, the optical isomers of propranolol displayed virtually identical stereospecific differences in soluble and membraneous preparations. However, the potency of various catecholamine agonists and the steepness of the displacement curves were greater in all solubilised preparations. Computer-assisted analysis of the displacement curves generated by the highly selective beta₁ antagonist atenolol and the beta₂ antagonist ICI 118.551, revealed the co-presence of beta₁ and beta₂ adrenoceptors in solubilised rabbit lung preparations. Furthermore, soluble beta₁ adrenoceptors appear to be much more labile at 22°C than soluble beta₂ adrenoceptors, providing support for the concept that these receptor subtypes are separate entities.     

Comparability of Mixed IC50 Data – A Statistical Analysis

The biochemical half maximal inhibitory concentration (IC₅₀) is the most commonly used metric for on-target activity in lead optimization. It is used to guide lead optimization, build large-scale chemogenomics analysis, off-target activity and toxicity models based on public data. However, the use of public biochemical IC₅₀ data is problematic, because they are assay specific and comparable only under certain conditions. For large scale analysis it is not feasible to check each data entry manually and it is very tempting to mix all available IC₅₀ values from public database even if assay information is not reported. As previously reported for K_i database analysis, we first analyzed the types of errors, the redundancy and the variability that can be found in ChEMBL IC₅₀ database. For assessing the variability of IC₅₀ data independently measured in two different labs at least ten IC₅₀ data for identical protein-ligand systems against the same target were searched in ChEMBL. As a not sufficient number of cases of this type are available, the variability of IC₅₀ data was assessed by comparing all pairs of independent IC₅₀ measurements on identical protein-ligand systems. The standard deviation of IC₅₀ data is only 25% larger than the standard deviation of K_i data, suggesting that mixing IC₅₀ data from different assays, even not knowing assay conditions details, only adds a moderate amount of noise to the overall data. The standard deviation of public ChEMBL IC₅₀ data, as expected, resulted greater than the standard deviation of in-house intra-laboratory/inter-day IC₅₀ data. Augmenting mixed public IC₅₀ data by public K_i data does not deteriorate the quality of the mixed IC₅₀ data, if the K_i is corrected by an offset. For a broad dataset such as ChEMBL database a K_i- IC₅₀ conversion factor of 2 was found to be the most reasonable.     

Synthesis,biological evaluation and molecular docking studies of new amides of 4-bromothiocolchicine as anticancer agents

Colchicine is the major alkaloid isolated from the plant Colchicum autumnale, which shows strong therapeutic effects towards different types of cancer. However, due to the toxicity of colchicine towards normal cells its application is limited. To address this issue we synthesized a series of seven triple-modified 4-bromothiocolchicine analogues with amide moieties. These novel derivatives were active in the nanomolar range against several different cancer cell lines and primary acute lymphoblastic leukemia cells, specifically compounds: 5–9 against primary ALL-5 (IC₅₀ = 5.3–14 nM), 5, 7–9 against A549 (IC₅₀ = 10 nM), 5, 7–9 against MCF-7 (IC₅₀ = 11 nM), 5–9 against LoVo (IC₅₀ = 7–12 nM), and 5, 7–9 against LoVo/DX (IC₅₀ = 48–87 nM). These IC₅₀ values were lower than those obtained for unmodified colchicine and common anticancer drugs such as doxorubicin and cisplatin. Further studies revealed that colchicine and selected analogues induced characteristics of apoptotic cell death but manifested their effects in different phases of the cell cycle in MCF-7 versus ALL-5 cells. Specifically, while colchicine and the studied derivatives arrested MCF-7 cells in mitosis, very little mitotically arrested ALL-5 cells were observed, suggesting effects were manifest instead in interphase. We also developed an in silico model of the mode of binding of these compounds to their primary target, β-tubulin. We conducted a correlation analysis (linear regression) between the calculated binding energies of colchicine derivatives and their anti-proliferative activity, and determined that the obtained correlation coefficients strongly depend on the type of cells used.     

Discovery and optimization of 4-oxo-2-thioxo-thiazolidinones as NOD-like receptor (NLR) family,pyrin domain-containing protein 3 (NLRP3) inhibitors

Aberrant activation of NLRP3 inflammasome is present in a subset of acute and chronic inflammatory diseases. The NLRP3 inflammasome has been recognized as an attractive therapeutic target for developing novel and specific anti-inflammatory inhibitors. Cellular structure-activity relationship-guided optimization resulted in the identification of 4-oxo-2-thioxo-thiazolidinone derivative 9 as a selective and direct small-molecule inhibitor of NLRP3 with IC₅₀ of 2.4 μM, possessing favorable ex vivo and in vivo pharmacokinetic properties. Compound 9 may represent a lead for the development of anti-inflammatory therapeutics for treating NLRP3-driven diseases.