Effects of dextromethorphan as add-on to sitagliptin on blood glucose and serum insulin concentrations in individuals with type 2 diabetes mellitus: a randomized, placebo-controlled, double-blinded, multiple crossover, single-dose clinical trial.

In this clinical trial, we investigated the blood glucose (BG)-lowering effects of 30, 60 and 90 mg dextromethorphan (DXM) as well as 100 mg sitagliptin alone versus combinations of DXM and sitagliptin during an oral glucose tolerance test (OGTT) in 20 men with T2DM. The combination of 60 mg DXM plus 100 mg sitagliptin was observed to have the strongest effect in the OGTT. It lowered maximum BG concentrations and increased the baseline-adjusted area under the curve for serum insulin concentrations in the first 30 min of the OGTT (mean ± standard deviation 240 ± 47 mg/dl and 8.1 ± 6.1 mU/l/h, respectively) to a significantly larger extent than did 100 mg sitagliptin alone (254 ± 50 mg/dl and 5.8 ± 2.5 mU/l/h, respectively; p < 0.05) and placebo (272 ± 49 mg/dl and 3.9 ± 3.0 mU/l/h, respectively; p < 0.001). All study drugs were well tolerated, alone and in combination, without serious adverse events or hypoglycaemia. Long-term clinical trials are now warranted to investigate the potential of the combination of 30 or 60 mg DXM and dipeptidyl peptidase-4 inhibitors in the treatment of individuals with T2DM, in particular as preclinical studies have identified the ß-cell protective properties of DXM.

Characterization of the gene encoding alpha-sarcin, a ribosome-inactivating protein secreted by Aspergillus giganteus.

The filamentous fungus, Aspergillus giganteus, produces the extracellular ribosome-inactivating protein, alpha-sarcin (Sar). The structural gene (sar) encoding Sar was isolated and characterized by sequence analysis and expression in Aspergillus niger. It codes for a precursor of 177 amino acids containing a secretion signal sequence that is absent in the mature protein. The nucleotide sequence contains several typical features of fungal genes, including a short intron of 65 bp. The transcriptional and translational processing signals of the gene are functional in A. niger, but the yield of recombinant protein is low in comparison with the natural producer. A comparison of sar with the gene encoding restrictocin from Aspergillus restrictus revealed a high degree of similarity between both genes. The conservation of the aa sequence suggests that Sar-like proteins may confer a selective advantage to these fungi under certain environmental conditions.

A novel chimaeric derivative of saruplase, rscu-PA-40 kDA/Hir, binds to thrombin and exerts thrombus-specific fibrinolysis in arterial and venous thrombosis in dogs.

The chimaeric molecule rscu-PA-40 kDA/Hir (M23) comprises the kringle and protease domain of saruplase (rscu-PA) and a thrombin inhibitory domain fused to the C-terminus of the protease domain. The 27 amino cid long thrombin inhibitory domain contains a sequence directed to the active site of thrombin and a fragment from the C-terminal region of hirudin. 125I-radiolabelled M23 (0.03 microM) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01-10 microM) and hirudin (0.001-10 microM) concentration-dependently displaced 125I-M23 from its binding to thrombin. Saruplase (up to 10 microM) did not influence the thrombin binding of M23. The fibrinolytic properties of M23 and saruplase were compared in anaesthetized dogs with femoral artery and saphenous vein thrombosis. Under concomitant heparinization, the intravenous bolus injections of 1 mg/kg M23 or saruplase induced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M23-treated group. Time to reperfusion (23 +/- 4 vs 25 +/- 11 min) and maximal height of reperfusion blood flow (98 +/- 21 vs 108 +/- 15% of baseline flow) did not differ significantly between the treatment groups. The time course of the lysis of incorporated 125I-fibrin radioactivity in thrombosed saphenous-veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of 125I-fibrin in the venous thrombosis model was 91 +/- 1% in M23- and 88 +/- 5% in saruplase-treated animals. Plasma levels of fibrinogen were not influenced and alpha 2-antiplasmin levels were slightly reduced (-27 +/- 3%) after bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 +/- 19%) and alpha 2-antiplasmin (-75 +/- 11%) plasma levels. Template bleeding times virtually did not differ before (2.8 +/- 0.3 min) and 60 min after bolus injection of M23 (3.1 +/- 0.3 min), whereas treatment with saruplase resulted in a significant prolongation of template bleeding time from 2.6 +/- 0.2 min to 28 +/- 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis after intravenous bolus injection in dogs, causes much fewer haemostatic side effects than its parent molecule. The high thrombus-specific activity of M23 is tentatively attributed to its affinity to clot-bound thrombin.

Immunomodulation by thalidomide: systematic review of the literature and of unpublished observations.

Three decades of immunological investigations using thalidomide are reviewed. Both in vitro and in vivo investigations are in accordance with the clinical finding that thalidomide does not impede T-cell competence in the control of infection by mycobacteriae. The term immunosuppressant does not apply. The immunomodulatory effects of thalidomide are evident in a myriad of phenomenological changes, and a molecularly defined common denominator of these activities is not known at present. Critical assessment with the objective to account for the clinical activity of thalidomide in specific human diseases leads to a focus on effects of thalidomide on phagocytic leukocytes and endothelia. The former are responsive to thalidomide by modulation of cytokine synthesis in vitro and in vivo; this activity can be shown using monocyte-specific stimuli in peripheral blood mononuclear cells but also in other phagocytic cells like microglia. For technical reasons, endothelial cells have until now been tested primarily in vitro. However, there is solid evidence now from intravital microscopy that the induction of adhesivity in postcapillary venules by LPS is modulated by thalidomide. Altered surface antigen expression has been described on leukocytes obtained from humans and experimental animals treated with thalidomide, but convincing evidence is lacking for in vitro modulation of surface antigen expression on leukocytes (as opposed to the modulation of adhesion antigens on endothelial cells stimulated by LPS or exogenous TNF alpha in the presence of thalidomide). Therefore, in vivo redistribution is likely to account for some, if not all, changes in circulating leukocyte phenotypes. The immunopathological conditions most clearly responsive to thalidomide are vasculitic alterations of post-capillary venules either in the context of mycobacterial infection (in the case of erythema nodosum leprosum) or mucocutaneous aphths. In both instances (as in the majority of focal inflammatory lesions), leukocyte infiltration and cytokine responses, in particular TNF alpha, are present. Thalidomide acts clinically not only by palliation of existing lesions but also by prevention of recurrence. The mechanism operates in skin, mucosa and parts of the nervous system and is most readily explained by synergism of TNF alpha modulation and a separate point of action on leukocyte migration patterns.

Agonistic effects of the opioid buprenorphine on the nociceptin/OFQ receptor.

The nociceptin/orphanin FQ (N/OFQ) receptor (e.g. the human ortholog ORL1) has been shown to be pharmacologically distinct from classic opioid receptors. Recently, we have identified buprenorphine as a full ORL1 agonist using a reporter gene assay. For further functional analysis, buprenorphine's effects on ORL1 receptors were investigated using a K(+) channel (GIRK1) assay in Xenopus oocytes and GTPgammaS assay in CHO-K1 membrane preparations. In both assays, buprenorphine behaved as a partial agonist compared to nociceptin itself. The N/OFQ agonism of buprenorphine might contribute to actions of buprenorphine in pain models in vivo beside its mu- or kappa-opioid receptor mediated effects.

Trichloroethanol inhibits ATP-induced membrane currents in cultured HEK 293-hP2X3 cells.

Membrane currents in response to the application of alpha, beta-methylene ATP (alpha,beta-meATP) were recorded by the whole-cell patch-clamp technique in human embryonic kidney 293 cells transfected with the human P2X3 receptor (HEK 293-hP2X3 cells). Trichloroethanol, the biologically active metabolite of chloral hydrate, but not ethanol itself concentration-dependently and reversibly inhibited the current responses. It was concluded that the reported analgesic effect of chloral hydrate may be due to the interruption of pain transmission in dorsal root ganglia expressing P2X3 receptors.

Thrombin inhibitory and clot-specific fibrinolytic activities of the urokinase variant, M23 (rscu-PA-40 kDa/Hir).

The recombinant bifunctional urokinase variant, M23 (rscu-PA-40 kDA/Hir), comprising the kringle and protease domain of single-chain urokinase-type plasminogen activator and a C-terminal fragment of hirudin in one single-chain molecule, was evaluated for its thrombin-inhibitory and fibrinolytic properties in vitro and in vivo. M23 inhibited thrombin-activated coagulation of human blood and thrombin-induced aggregation of human platelet rich plasma in a concentration-dependent manner. The ADP-induced aggregation of human platelet rich plasma was not influenced by M23. In contrast, recombinant single-chain urokinase-type plasminogen activator (saruplase) inhibited neither blood coagulation nor platelet rich plasma aggregation. M23 and saruplase both lysed radiolabelled human thrombi immersed in human plasma (Chandler Loop system) with equal potency. However, there was a significantly lower systemic generation of plasmin (measured as consumption of alpha 2-antiplasmin) by M23 compared to saruplase. In anaesthetized non-heparinized rabbits, experimental femoral artery thrombosis was treated with intravenous bolus injections of M23 or saruplase (6 mg/kg, each). Thrombolytic restoration of arterial blood perfusion was significantly higher in M23- than in saruplase-treated rabbits. Plasma fibrinogen concentrations were decreased markedly in saruplase-treated animals, but remained at significantly higher levels in M23-treated rabbits. In conclusion, the bifunctional molecule, M23, showed thrombin inhibitory and fibrinolytic properties in human in vitro systems and exerted superior thrombolytic effects to saruplase in rabbit femoral artery thrombosis. In vitro and in vivo data indicate that the fibrinolytic activity of M23 is highly clot-specific.

Amyloid beta peptides stimulate tissue-type plasminogen activator but not recombinant prourokinase.

Tissue-type plasminogen activator (rt-PA) and prourokinase (rscu-PA) have been tested with respect to the influence of amyloid beta peptides on plasminogen activation which was monitored by cleavage of the chromogenic plasmin substrate S-2251. It was shown that rt-PA is stimulated by amyloid beta peptides at concentrations of 10 micrograms/ml in contrast to prourokinase, which does not alter its catalytic properties in presence of amyloid beta peptides. The stimulation of rt-PA can be inhibited by tranexamic acid indicating a molecular mode of stimulation similar to the fibrin mediated stimulation of rt-PA.

Affinity, potency and efficacy of tramadol and its metabolites at the cloned human mu-opioid receptor.

The present study was conducted to characterise the centrally active analgesic drug tramadol hydrochloride [(1RS,2RS)-2-[(dimethyl-amino)-methyl]-1-(3-methoxyphenyl)-cyclohe xanol hydrochloride] and its metabolites M1, M2, M3, M4 and M5 at the cloned human mu-opioid receptor. Membranes from stably transfected Chinese hamster ovary (CHO) cells were used to determine the four parameters of the ligand-receptor interaction: the affinity of (+/-)-tramadol and its metabolites was determined by competitive inhibition of [3H]naloxone binding under high and low salt conditions. The agonist-induced stimulation of [35S]GTPgammaS binding permits the measurement of potency (EC50), efficacy (Emax = maximal stimulation) and relative intrinsic efficacy (effect as a function of receptor occupation). The metabolite (+)-M1 showed the highest affinity (Ki=3.4 nM) to the human mu-opioid receptor, followed by (+/-)-M5 (Ki=100 nM), (-)-M1 (Ki=240 nM) and (+/-)-tramadol (Ki=2.4 microM). The [35S]GTPgammaS binding assay revealed an agonistic activity for the metabolites (+)-M1, (-)-M1 and (+/-)-M5 with the following rank order of intrinsic efficacy: (+)-M1>(+/-)-M5>(-)-M1. The metabolites (+/-)-M2, (+/-)-M3 and (+/-)-M4 displayed only weak affinity (Ki> 10 microM) and had no stimulatory effect on GTPgammaS binding. These data indicate that the metabolite (+)-M1 is responsible for the mu-opioid-derived analgesic effect.

Comparative study of tacrolimus and paclitaxel stent coating in the porcine coronary model.

BACKGROUND: Tacrolimus is a potent antiproliferative and immunosuppressive agent allowing for improved endothelial regeneration. The aim of our study was the preclinical evaluation of tacrolimus in a drug eluting nonerodable polymer stent system and its comparison with paclitaxel. METHODS AND RESULTS: A total of 40 domestic pigs and 10 mini-pigs underwent coronary stenting with a follow-up time between 6 hours and 3 months. Stents were implanted in coronary arteries with an overstretch ratio of 1.2. After 3 days, a 1.73 microg/mm(2) coating produced tacrolimus tissue levels of 20 mumol/l in the coronary artery wall. Effective tissue concentrations were sustained for 28 days. Based on histomorphometric analysis, tacrolimus stent treated vessels had a reduced extent of neointima formation compared with controls at 28 days (-51% compared to control) but not at 3 months. High dose paclitaxel stent coating (1.44 microg/mm(2)) was complicated by unexpected deaths of pigs and thrombotic stent occlusion at control angiography. Long-term porcine data showed no persistent inhibition of neointimal growth by paclitaxel and tacrolimus stent coating. CONCLUSIONS: Similar to paclitaxel, tacrolimus stent coating reduces neointimal proliferation in the porcine coronary model. However, dosing and long-term efficacy remains a critical issue in stent-based local drug delivery.

Molecular cloning, sequence analysis and expression of the gene encoding an antifungal-protein from Aspergillus giganteus.

The gene encoding the precursor of a small secretory protein with antifungal activity was isolated from A. giganteus and characterized by restriction mapping, hybridization and nucleotide sequencing. The promoter contains a typical TATA-box at a distance of 135 bp upstream of the open reading frame. The open reading frame is interrupted by two small introns with conserved splice sites. The precursor of the antifungal protein (AFP) consists of 94 amino acids and appears to be processed to the mature AFP of 51 amino acids by a two-step process. Transfer of the gene into A. niger yielded only transformants with a very low expression level, probably because high-expression transformants were counterselected by the antifungal activity of the recombinant protein.

Transformation of Aspergillus giganteus to hygromycin B resistance.

A wild strain of A. giganteus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of A. nidulans sequences. Stable transformants arose by heterogenous integration, mainly of tandem repeats of vector DNA at various sites in the host genome. Between 6 and 30 resistant colonies were obtained per microgram DNA per 3 x 10(3) viable protoplasts. Vector DNA could be recovered by transformation of Escherichia coli with undigested genomic DNA from Aspergillus giganteus transformants.

5'-Substituted thalidomide analogs as modulators of TNF-alpha.

The synthesis of 5'-substituted thalidomide analogs is described. The amino acids 2 necessary to synthesize the target compounds were prepared by Michael reaction. Condensation of 2 with phthalic anhydrides followed by reaction with urea yielded 4 as diastereomeric mixtures. Furthermore glutethimide (5) was brominated by an improved method and the resulting compound 6 was reacted in several steps with sodium azide, hydrogen, and phthalic anhydride to give 8. In a similar manner, 6 was reacted with sodium azide and various phthalic anhydrides to give 9, 10, and 11. All final compounds were tested in vitro for their inhibitory activity on the release of TNF-alpha, using stimulated peripheral mononuclear blood cells (PBMCs). Compounds with an additional aromatic substituent in position 5' of the thalidomide molecule were more active than thalidomide. Compound 11 was able to reduce increased levels of IL-2 in vitro.

A strong thrombin-inhibitory prourokinase derivative with sequence elements from hirudin and the human thrombin receptor.

In order to design plasminogen activators with improved thrombolytic properties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin-inhibitory moiety itself comprises four elements: linker 1, a motif directed to thrombin's active site, linker 2 and a fragment of hirudin which binds to the fibrinogen-recognition site of thrombin. In order to improve further the anticoagulative activity, the thrombin-inhibitory domain was modified by substituting linker 2. Introduction of a linker (FLLRNP) from the human thrombin receptor conferred about a 10-fold increase in anticoagulative activity in protein M37 compared with the parent molecule M23 carrying an aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the Ki value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the thrombin receptor which may interact with one of two glutamic acid residues located at the exit of the thrombin substrate binding pocket. This explanation is supported by the observation that another chimera (M35) carrying a linker sequence with two acidic residues has relatively weak thrombin-inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential.

The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin.

Enantioselective inhibition of TNF-alpha release by thalidomide and thalidomide-analogues.

The question whether the immunomodulating activity of rac-thalidomide resides in either the (-)-(S)- or the (+)-(R)-enantiomer was addressed by synthesis and separation of pure enantiomers of thalidomide analogues which carry a methyl-group at the asymmetric carbon atom and are thus prevented from racemization. The effect of the pure enantiomers of the thalidomide-analogues and also of the enantiomers of thalidomide on release of TNF-alpha was tested in vitro by using stimulated peripheral mononuclear blood cells. Both enantiomers of thalidomide inhibited the release of TNF-alpha equally well at low concentrations (5 and 12.5 micrograms/ml) but at higher concentrations (25 and 50 micrograms 50 micrograms/ml) there was a weak but statistically significant selectivity towards the (-)-(S)-enantiomer. In the case of the configuration-stable thalidomide-analogues there was a very pronounced and statistically significant enantioselectivity towards the (S)-form even at lower concentrations (> or = 5 micrograms/ml). The (S)-enantiomers of the thalidomide-analogues differed in their inhibitory potency from (-)-(S)-thalidomide suggesting that the introduction of the methyl-group increases the TNF-alpha-inhibitory activity while the reduction of one of the carbonyl-functions in the glutarimide-moiety to a methylene-group decreases activity. The effect of these small molecular alterations on activity and the enantioselectivity towards the (S)-enantiomers may indicate that thalidomide and its analogues directly interact with one or several cellular target-proteins.

Isolation and physical properties of the DNA-directed RNA polymerase from Thermus thermophilus HB8.

The DNA-directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method. The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for beta and beta', 40 kDa for alpha and 92 kDa for sigma. The RNA polymerase is active at elevated temperatures (65 degrees C). Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity. The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T. thermophilus templates whose in vivo starts of transcription were characterized by nuclease S1 mapping.

Synthesis and immunological activity of water-soluble thalidomide prodrugs.

A series of new water-soluble thalidomide prodrugs was prepared. All compounds were derivatized on the nitrogen of the glutarimide ring. Esters of natural amino acids and succinic acid derivatives have been introduced by reaction with the hydroxymethyl thalidomide 2. Nicotinic acid derivatives were prepared from halomethyl derivatives. Additionally, a methoxymethyl derivative and a carboxymethyl derivative were prepared directly from thalidomide. Most compounds showed a very large increase in water solubility compared to thalidomide itself (0.012mg/mL). The amorphous hydrochlorides of the N-methylalanine ester 8, valine ester 9, and glycylglycine ester 10, respectively, were the most soluble compounds showing solubility greater than 300mg/mL, which equals an increase greater than 15,000-fold. The lipophilicity of the prodrugs has been determined by their HPLC capacity factors k'. The stability of selected compounds was determined. The hydrolysis rates follow pseudo-first order kinetics. In order to assess the immunological activity, the prodrugs were tested using tumor necrosis factor-alpha and interleukin-2 inhibition assays. Selected compounds were additionally investigated on their abililty to inhibit the local Shwartzman reaction, an assay to determine the vascular permeability. The prodrugs retained high effectiveness in the inhibition of TNF-alpha release. Our results indicated that the more stable prodrugs exhibited higher activity in the immunological assays. Some compounds showed higher activity than thalidomide itself, suggesting a high affine binding to the pharmacophore. In conclusion, the prodrugs exhibited high water solubility and high activity and might therefore be used in therapeutic applications.