A pooled analysis of randomized, controlled, phase 3 trials investigating the efficacy and safety of a novel, fixed dose calcipotriene and betamethasone dipropionate cream for the topical treatment of plaque psoriasis.

BACKGROUND: Plaque psoriasis is a common, chronic and relapsing inflammatory skin disease clinically characterized by erythema and scaling desquamation. As over 90% of psoriasis patients benefit from topical therapies, local treatments continue to play an eminent role in management strategies. One such topical treatment is the fixed dose combination of calcipotriol (CAL) and betamethasone dipropionate (BDP). OBJECTIVES: Pooled analysis of two different phase 3 clinical trails to compare superiority regarding efficacy, safety and quality of life (QoL) between CAL/BDP PAD-cream and CAL/BDP TS. METHODS: The data from two phase 3, multicentre, randomized, investigator-blind, active and vehicle-controlled trials enrolling patients with psoriasis were pooled and analysed. Investigational products included a CAL/BDP cream based on PAD™ Technology (PAD-cream) designed for high skin penetration and increased patient preference, an active control (marketed CAL/BDP topical suspension/gel, in the following abbreviated as CAL/BDP TS) and cream vehicle, which were applied once daily for 8 weeks. RESULTS: Efficacy and safety of the novel CAL/BDP PAD-cream formulation for the topical treatment of psoriasis demonstrated superiority for all efficacy end points after 8 weeks of treatment. PGA treatment success for CAL/BDP PAD-cream (43.2%) was greater than CAL/BDP TS (31.9%; P < 0.0001), the mean per cent reduction in mPASI for CAL/BDP PAD-cream was 64.6% compared to 56.4% for CAL/BDP TS (P < 0.0001) and DLQI 0/1 was obtained by 43.8% in the CAL/BDP PAD-cream group versus 34.2% in the CAL/BDP TS group (P = 0.0005). There was no adverse drug reaction reported with a frequency of >1%, associated with the CAL/BDP PAD-cream. CONCLUSIONS: The novel fixed dose combination CAL/BDP PAD-cream offers greater efficacy, superior patient QoL and equivalent favourable safety for the topical treatment of psoriasis, in comparison to the currently available topical suspension/gel.

A randomized, phase IIa exploratory trial to assess the safety and preliminary efficacy of LEO 43204 in patients with actinic keratosis.

BACKGROUND: LEO 43204 is a novel ingenol derivative in development for the treatment of actinic keratosis. OBJECTIVES: To compare the safety and preliminary efficacy of three doses of LEO 43204 with ingenol mebutate in actinic keratoses (AKs). METHODS: Patients with at least three visible, discrete, nonkeratotic AKs on four separate selected treatment areas on the forearms received LEO 43204 gel (0·025%, 0·05% and 0·075%) and ingenol mebutate 0·05% gel, by investigator-blinded, randomized allocation, for 2 consecutive days. Patients were assessed at 8 weeks. Primary outcomes included maximum composite local skin response (LSR) score and adverse events (AEs). Secondary outcomes included a reduction in the number of visible AKs. RESULTS: Forty patients completed the trial. For all treatments, mean LSR scores peaked at week 1, and were below baseline by week 8. Mean maximum composite LSR scores for LEO 43204 0·025%, 0·05% and 0·075% were 9·2 (Dunnett adjusted P = 0·02), 10·1 (Dunnett adjusted P = 0·90) and 11·2 (Dunnett adjusted P < 0·01), respectively, vs. ingenol mebutate 0·05% gel (10·0). The most frequent AEs across all treatments were application site pruritus, burning sensation and tenderness. Mean reduction in the number of AKs was comparable for ingenol mebutate and the two lowest doses of LEO 43204 (71·9-73·1%), but LEO 43204 0·075% gave a significantly larger reduction (81·8%; Dunnett adjusted P = 0·04). CONCLUSIONS: LEO 43204 had a similar safety profile to ingenol mebutate and a dose-response relationship for LSRs was demonstrated. The highest LEO 43204 dose (0·075%) significantly reduced the AK count when compared with ingenol mebutate.

Ethanol metabolism and lipid synthesis by isolated liver cells from fed rats.

1. The fatty acid synthesis in isolated liver cells from fed rats was studied with tritiated water as the radioactive precursor. The cells incorporated 3H20 at a rate of 1.26 mumol per min per g packed cells. 2. Addition of ethanol caused a 20% decrease in the incorporation of tritium into fatty acids. The decrease was correlated to the increase in the NAD-redox level. Probably, the decreased tritium incorporation into fatty acids during ethanol metabolism is due to a decrease in the specific activity of the NADPH used for the synthesis of fatty acids, rather than to a real inhibition of the fatty acid synthesis. 3. Ethanol oxidation via NADPH-consuming pathways and ethanol per se at a concentration of 80 mM had no effect upon the incorporation of tritium into fatty acids. 4. Fructose in a concentration of 15 mM inhibited the fatty acid synthesis by 75%, and this inhibition was further augmented by ethanol. 5. The ioslated rat liver cells oxidized ethanol at a rate of 2.72, 2.93 and 3.48 mumol per min per g packed cells at 5, 20 and 80 mM ethanol, respectively. Fructose had no effect upon ethanol oxidation neither at low nor at high concentrations of ethanol. 6. Ethanol oxidation via the non alcohol dehydrogenase pathway(s) may involve a transfer of reducing equivalents from mitochondrial NADH to cyctosolic NADP+ as judged from measurements of metabolite levels. This conclusion is supported by determinations of 14C yield in glucose from [1-14C] ethanol, and the results are taken as evidence for the presence of hydrogen shuttle activity during metabolism of ethanol, catalyzed by the NAD-dependent alcohol dehydrogenase. A metabolic scheme is proposed to account for the observed changes at low and high concentrations of ethanol.

Biological background subtraction improves immunoscintigraphy by subsequent injection of antigen.

We have developed an 111In-labeled antibody for in vivo use directed against tissue plasminogen activator demonstrating focal fibrinolytic activity. However, a major problem in immunoscintigraphy is the low signal-to-noise ratio due to circulating antibody. The hepatic clearance of t-PA is very rapid. The effect of a subsequent injection of a small amount of t-PA shortly after the antibody administration to increase the blood clearance rate of the formed antigen-antibody complexes was examined in six rabbits. More than 99% of the circulating antigen-antibody complexes were eliminated by the liver within 10 min. This technique could make immunoscintigraphy a first line diagnostic tool in acute medicine including imaging of thromboembolic lesions in organs with high blood volumes such as the lungs, the heart, and the brain.

A specific immunologic assay for functional plasminogen activator inhibitor 1 in plasma--standardized measurements of the inhibitor and related parameters in patients with venous thromboembolic disease.

A new assay for functional plasminogen activator inhibitor 1 (PAI-1) in plasma was developed. The assay is based on the quantitative conversion of PAI-1 to urokinase-type plasminogen activator (u-PA)-PAI-1 complex the concentration of which is then determined by an ELISA employing monoclonal anti-PAI-1 as catching antibody and monoclonal anti-u-PA as detecting antibody. The assay exhibits high sensitivity, specificity, accuracy, and precision. The level of functional PAI-1, tissue-type plasminogen activator (t-PA) activity and t-PA-PAI-1 complex was measured in normal subjects and in patients with venous thromboembolism in a silent phase. Blood collection procedures and calibration of the respective assays were rigorously standardized. It was found that the patients had a decreased fibrinolytic capacity. This could be ascribed to high plasma levels of PAI-1. The release of t-PA during venous occlusion of an arm for 10 min expressed as the increase in t-PA + t-PA-PAI-1 complex exhibited great variation and no significant difference could be demonstrated between the patients with a thrombotic tendency and the normal subjects.

Plasminogen activator inhibitor-1 is the primary inhibitor of tissue-type plasminogen activator in pregnancy plasma.

The aim of the present work was to clarify to what extent plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) contribute to the increase in plasma inhibition of tissue-type plasminogen activator (t-PA) observed during pregnancy. It was demonstrated that a monoclonal antibody against PAI-1 almost completely quenched inhibition of single-chain t-PA and most of the inhibition of two-chain t-PA in plasma during the third trimester of pregnancy. The remaining inhibition of two-chain t-PA was to a great extent abolished by a PAI-2 antibody. The second order rate constant (k1) for inhibition of single-chain t-PA by the inhibitor neutralized by the PAI-1 antibody was about 4.8.10(6) M-1.s-1. The conversion of single-chain t-PA to the two-chain form increased the reaction rate with the inhibitor about 3-fold. These kinetic data are comparable with those obtained with PAI-1 in non-pregnancy plasma or with purified PAI-1. From the above results it is concluded that PAI-1 is the primary inhibitor of both single-chain and two-chain t-PA and that PAI-2 is the secondary inhibitor of two-chain t-PA in pregnancy plasma. The concentration of reactive PAI-1 versus gestation age was assayed in plasma from 6 women by binding of PAI-1 to 125I-labelled single-chain t-PA followed by quantitation of the labelled t-PA-PAI-1 complex after separation by SDS-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological relationship between the fast-acting plasminogen activator inhibitors from plasma, blood platelets and endothelial cells demonstrated with a monoclonal antibody against an inhibitor from placenta.

Two plasminogen activator inhibitors (I and II) were demonstrated in human placenta. The complex between inhibitor I and tissue-type plasminogen activator was purified by immunoadsorption to solid-phase anti-activator antibodies. The purified complex (Mr 95.000) was used for immunization of mice and subsequent production of monoclonal antibodies. One antibody (F37), which reacted with both free and complex-bound inhibitor I, was used for further study by a method involving binding of the antibody to protein A-Sepharose, immunoadsorption of antigen and analysis of the resulting supernatant by SDS-polyacrylamide gel electrophoresis and enzymography. The analysis showed that F37 reacted with the fast-acting plasminogen activator inhibitors recently demonstrated in plasma, blood platelets and endothelial cells, indicating that these inhibitors and inhibitor I share a common epitope. Inhibitor II did not react with F37. Inhibitor II is identical to the placenta inhibitor previously described by others. It reacted selectively with polyclonal antibodies against that inhibitor.

A sensitive solid-phase immunosorbent assay for tissue-type plasminogen activator activity in plasma using trinitrobenzoylated poly-D-lysine as a stimulator for plasminogen activation.

A sensitive, specific and precise immunosorbent assay for tissue-type plasminogen activator (t-PA) activity in plasma was developed. It measured the single-chain and the two-chain forms of t-PA with equal sensitivity. The assay involved (I) coating of wells in microtiter plates with a monoclonal antibody directed towards an epitope on t-PA apart from the catalytic active site, (II) binding of t-PA to the solid-phase antibody, (III) activation of plasminogen by antibody-bound t-PA in the presence of a new potent stimulator, trinitrobenzyl alkylated poly-D-lysine (TNB-poly-D-lysine) and measurement of plasmin activity with D-Val-Leu-Lys-pNA. Plasma samples were acid-treated and diluted 80 times in order to minimize the inhibitory effect of plasma on the assays. The assay could be performed within one working day with precoated microtiter plates. The sensitivity of the assay for t-PA in plasma was 1 pM (approximately 70 ng/l). The recoveries of single-chain and two-chain t-PA added to plasma was 97-104%. The intraassay coefficient of variation was 3.4-5.1% and the interassay coefficient of variation was 7.8-18%. Resting values of t-PA in plasma for 42 healthy subjects ranged between 0 and 30 pM (median: 4.1 pM). The values after 10 min venous occlusion ranged between 1.2 and 520 pM (median: 100 pM). The t-PA concentrations determined by the immunosorbent assay correlated well with euglobulin clot lysis time measurements (r = 0.940).

Modulation of metabolism through transcriptional control has created new treatment opportunities for type 2 diabetes.

The discovery of the important metabolic and physiological role played by a family of transcription factors, the peroxisome proliferator activated receptors (PPAR), has opened up for a new understanding of the mode of action for the lipid lowering drugs known as fibrates and for the new glucose lowering compounds described as insulin sensitizers. Both of these classes of compounds have demonstrated significant efficacy in both animal models of the metabolic derangements characteristic for type 2 diabetes and in human clinical studies. The recognition of the role of these drugs as ligands for PPAR transcription factors and the development of new molecular and cellular tools to select and characterise new PPAR selective compounds will open up for the development of even better new drug candidates for the treatment of metabolic disorders associated with type 2 diabetes. With the combined strength of new transcriptional mapping technologies developed in the field of molecular biology, such as differential mRNA display and DNA microarray hybridisations, it will be possible to perform a detailed molecular characterisation of the transcriptional events involved in drug actions in cellular and tissue systems, and information gathered from such types of analysis will lead to an enormous amount of data, from which detailed knowledge of drug actions at the gene regulatory level will emerge.

Immunochemical determination of acetylcholinesterase in amniotic fluid--an evaluation of eleven monoclonal antibodies.

Eleven monoclonal antibodies and a polyclonal rabbit antiserum were evaluated with respect to reactivity with acetylcholinesterase (AChE, EC 3.1.1.7) from erythrocytes and brain. Employing our enzyme antigen immunoassay for AChE five selected antibodies were evaluated with regard to their clinical usefulness in the prenatal diagnosis of neural tube defects (NTD). Of these, one antibody preferentially bound the enzyme from human brain, and discerned better than the others pathological samples (anencephaly, spina bifida and encephalocele) from normal ones. With this antibody no false positive values were obtained even if amniotic fluid samples were blood contaminated.

Diabetic late complications: will aldose reductase inhibitors or inhibitors of advanced glycosylation endproduct formation hold promise?

Patients suffering from the severe complications associated with both insulin- (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM): nephropathy, retinopathy, neuropathy, and atherosclerosis are still largely left without a prospect of an efficient treatment. This is the case even if it has been assumed for decades and now finally proved by the results from the Diabetes Control and Complications Trial (DCCT) that hyperglycemia is the single main cause of these complications. Improved glycemic control as a result of intensive insulin treatment has the potential to reduce the incidence and progression of complications, but implementation and monitoring of improved glycemic control in all groups of IDDM and NIDDM patients in different communities will be difficult and expensive. Results from the recently terminated DCCT have shown that even with intensive insulin treatment, there will be a significant burden of complications on the diabetic population. It will, therefore, still be of immense importance for the long-term quality of life for the diabetic patient that additional possibilities are developed for prevention and intervention against diabetic complications. Almost two decades of research, animal model testing, and clinical trials have been conducted on various efficient aldose reductase inhibitors. Now the concept of inhibition of formation of advanced glycosylation endproducts on proteins and lipids resulting from extra- and intracellular hyperglycemia is entering the scene as an alternative or perhaps supplementary approach to reduce the occurrence of diabetic complications. An overview of the results from these two fields of research and associated drug-development programs will be presented along with thoughts on possible future developments.

Hypofractionation vs conventional radiation therapy for newly diagnosed diffuse intrinsic pontine glioma: a matched-cohort analysis.

PURPOSE: Despite conventional radiation therapy, 54 Gy in single doses of 1.8 Gy (54/1.8 Gy) over 6 weeks, most children with diffuse intrinsic pontine glioma (DIPG) will die within 1 year after diagnosis. To reduce patient burden, we investigated the role of hypofractionation radiation therapy given over 3 to 4 weeks. A 1:1 matched-cohort analysis with conventional radiation therapy was performed to assess response and survival. METHODS AND MATERIALS: Twenty-seven children, aged 3 to 14, were treated according to 1 of 2 hypofractionation regimens over 3 to 4 weeks (39/3 Gy, n=16 or 44.8/2.8 Gy, n=11). All patients had symptoms for ≤3 months, ≥2 signs of the neurologic triad (cranial nerve deficit, ataxia, long tract signs), and characteristic features of DIPG on magnetic resonance imaging. Twenty-seven patients fulfilling the same diagnostic criteria and receiving at least 50/1.8 to 2.0 Gy were eligible for the matched-cohort analysis. RESULTS: With hypofractionation radiation therapy, the overall survival at 6, 9, and 12 months was 74%, 44%, and 22%, respectively. Progression-free survival at 3, 6, and 9 months was 77%, 43%, and 12%, respectively. Temporary discontinuation of steroids was observed in 21 of 27 (78%) patients. No significant difference in median overall survival (9.0 vs 9.4 months; P=.84) and time to progression (5.0 vs 7.6 months; P=.24) was observed between hypofractionation vs conventional radiation therapy, respectively. CONCLUSIONS: For patients with newly diagnosed DIPG, a hypofractionation regimen, given over 3 to 4 weeks, offers equal overall survival with less treatment burden compared with a conventional regimen of 6 weeks.

New procedure for extraction of ammonium from natural waters for N isotopic ratio determinations.

A new method for extracting ammonium from natural waters for N isotopic ratio determination is described. The method employs the conversion of the ammonium nitrogen into indophenol, which is then concentrated onto an octadecylsilane column. The method shows accuracy and precision comparable to those of other methods described in the literature. Some results from field experiments on the Swedish west coast are presented.

Native and degraded fibronectin: new immunological methods for distinction.

Two methods for quantitative determination of the high molecular weight glycoprotein, fibronectin, have been developed. Both methods are based on enzyme-linked immunoadsorbent (ELISA) techniques. In the first of the methods an antibody against fibronectin is used to trap the antigen. This double antibody technique can detect a slight decrease in the concentration of fibronectin stored for 5 days compared to the amount of fibronectin in the freshly purified preparation. In the second method, gelatin which is known to bind specifically to fibronectin, is used to catch fibronectin. By this method less than 1% of the fibronectin present in a freshly prepared preparation is measured after storage for 5 days. The results obtained with the two methods applied on a freshly prepared and a stored fibronectin are in agreement with sodium dodecylsulphate polyacrylamide gel electrophoresis followed by immunoblotting before and after gelatin-Sepharose adsorption. These techniques demonstrate that all the freshly prepared fibronectin adsorbs to gelatin-Sepharose, while stored fibronectin, which is broken down to numerous peptides, still reacts with the fibronectin antibody, but does not adsorb to gelatin-Sepharose. The two ELISA techniques were applied on amniotic fluid, cerebrospinal fluid and urine. The results indicated significant degradation of fibronectin in urine, and less degradation of fibronectin in amniotic and spinal fluid.

Complement C5a receptor antagonism by protamine and poly-L-Arg on human leukocytes.

It is shown that protamine selectively and dose-dependently inhibits complement C5a-induced leukocyte responses such as histamine release from basophils, chemiluminescence and beta-glucuronidase release from neutrophils. Protamine produces parallel rightward displacements of the C5a dose-response curves. The inhibitory capacity of the polypeptide is reversible and disappears following repeated washing of exposed cells. In neutrophils poly-L-Arg similarly and specifically antagonizes C5a-induced chemiluminescence and enzyme release. This polymer alone, however, degranulates basophils and neutrophils, leading to histamine and enzyme release, respectively. It is concluded that on human neutrophils the arginine-rich polycations protamine and poly-L-Arg exhibit a competitive C5a receptor antagonism. In addition, protamine inhibits the C5a receptors on basophils. It is hypothesized that molecular conformations of the arginine-rich polycations might bind reversibly to, and block negatively charged groups at the C5a-receptor sites.

Purification of Bordetella pertussis antigens using monoclonal antibodies.

Eleven monoclonal antibodies were produced using whole Bordetella pertussis cells as the immunizing antigen. All monoclonal antibodies reacted with components of Bordetella pertussis, as visualized in immunoblotting of SDS polyacrylamide gels. Selected antibodies were coupled to Sepharose columns and used for isolation of the corresponding antigen. In all cases complete accordance was found between SDS polyacrylamide gel electrophoresis of the eluted antigen and the bands found in immunoblotting of the original extract stained with the respective monoclonal antibody. One major problem in the interpretation of the results was the finding that some of the monoclonal antibodies stained a number of bands in immunoblottings of crude B.pertussis extract. This phenomenon was shown to be caused by proteolytic degradation of the antigens, since prior addition of protease inhibitors to the extract resulted in the staining of only one band. The monoclonal antibodies showed different reactivity patterns with various strains of B.pertussis, B.parapertussis, B.bronchiseptica and less closely related bacteria. Two of the antibodies were strictly specific for B.pertussis.