Caplacizumab reduces the frequency of major thromboembolic events, exacerbations and death in patients with acquired thrombotic thrombocytopenic purpura.

Essentials Acquired thrombotic thrombocytopenic purpura (aTTP) is linked with significant morbidity/mortality. Caplacizumab's effect on major thromboembolic (TE) events, exacerbations and death was studied. Fewer caplacizumab-treated patients had a major TE event, an exacerbation, or died versus placebo. Caplacizumab has the potential to reduce the acute morbidity and mortality associated with aTTP. SUMMARY: Background Acquired thrombotic thrombocytopenic purpura (aTTP) is a life-threatening autoimmune thrombotic microangiopathy. In spite of treatment with plasma exchange and immunosuppression, patients remain at risk for thrombotic complications, exacerbations, and death. In the phase II TITAN study, treatment with caplacizumab, an anti-von Willebrand factor Nanobody® was shown to reduce the time to confirmed platelet count normalization and exacerbations during treatment. Objective The clinical benefit of caplacizumab was further investigated in a post hoc analysis of the incidence of major thromboembolic events and exacerbations during the study drug treatment period and thrombotic thrombocytopenic purpura-related death during the study. Methods The Standardized Medical Dictionary for Regulatory Activities (MedDRA) Query (SMQ) for 'embolic and thrombotic events' was run to investigate the occurrence of major thromboembolic events and exacerbations in the safety population of the TITAN study, which consisted of 72 patients, of whom 35 received caplacizumab and 37 received placebo. Results Four events (one pulmonary embolism and three aTTP exacerbations) were reported in four patients in the caplacizumab group, and 20 such events were reported in 14 patients in the placebo group (two acute myocardial infarctions, one ischemic stroke, one hemorrhagic stroke, one pulmonary embolism, one deep vein thrombosis, one venous thrombosis, and 13 aTTP exacerbations). Two of the placebo-treated patients died from aTTP during the study. Conclusion In total, 11.4% of caplacizumab-treated patients and 43.2% of placebo-treated patients experienced one or more major thromboembolic events, experienced an exacerbation, or died. This analysis shows the potential for caplacizumab to reduce the risk of major thromboembolic morbidities and mortality associated with aTTP.

A new approach in the prediction of the dissolution behavior of suspended particles by means of their particle size distribution.

Though various attempts have been made in literature to model the particle size distribution of an active pharmaceutical ingredient (API) in function of the required release profile of the pharmaceutical product, so far one has not succeeded to develop a universal approach in the correlation of particle size distribution and in vitro dissolution data. In this publication, a new approach is presented on the use of particle size distribution data in the prediction of the in vitro dissolution profile of a suspension formulation. For this purpose, various theoretical experiments were done simply on paper and based on the Noyes-Whitney [A.A. Noyes, W.R. Whitney, J. Am. Chem. Soc. 19 (1897) 930-934] equation, the normalized dissolution profiles of various imaginary size distributions were calculated. For each size distribution, its weighted mean diameters were then calculated. Based on these theoretical data, a model could be developed which scientifically explains the dissolution profile of a suspension in function of its volume-weighted mean particle size (D[4, 3]). The applicability of this correlation model could experimentally be confirmed by evaluation of laser diffraction and in vitro dissolution data as they were obtained for different batches of a suspension formulation. This new approach in the correlation between particle size and dissolution may be an important analytical tool in the engineering of the particle size distribution of drug substance, and more precisely monitoring the D[4, 3] volume-weighted mean diameter may allow one to model the dissolution profile of a suspension formulation and thereby its in vivo release profile.

Laser diffraction and image analysis as a supportive analytical tool in the pharmaceutical development of immediate release direct compression formulations.

Immediate release direct compression tablet formulations require a strict control of the particle characteristics (i.e. particle size (distribution) and shape) of both the active pharmaceutical ingredient (API) and the excipients. In this publication, the development of a dry dispersion laser diffraction (LD) method has been outlined. With this method, the chemical development of an API meant for the manufacturing of an immediate release direct compression tablet formulation can be supported. Comparison with static image analysis (SIA) and scanning electron microscopy (SEM) data often shows laser diffraction to generate different size data. However, since LD is fast and frequently shows an adequate precision over a wide particle size range, the technique is still considered as a valuable analytical tool in the screening of the particle size distribution of API batches. In the future, automated (static) image analysis and dynamic image analysis are believed to become more and more important, since these techniques will allow the fast analysis of large amounts of particles with a minimum intervention of the operator.

Fractional flow reserve in patients with prior myocardial infarction.

BACKGROUND: Fractional flow reserve (FFR), an index of coronary stenosis severity, can be calculated from the ratio of hyperemic distal to proximal coronary pressure. An FFR value of 0.75 can distinguish patients with normal and abnormal noninvasive stress testing in case of normal left ventricular function. The present study aimed at investigating the value of FFR in patients with a prior myocardial infarction. Methods and Results-- In 57 patients who had sustained a myocardial infarction >/=6 days earlier, myocardial perfusion single photon emission scintigraphy (SPECT) imaging and FFR were obtained before and after angioplasty. The sensitivity and specificity of the 0.75 value of FFR to detect flow maldistribution at SPECT imaging were 82% and 87%. The concordance between the FFR and SPECT imaging was 85% (P<0.001). When only truly positive and truly negative SPECT imaging were considered, the corresponding values were 87%, 100%, and 94% (P<0.001). Patients with positive SPECT imaging before angioplasty had a significantly lower FFR than patients with negative SPECT imaging (0.52+/-0.18 versus 0.67+/-0.16, P=0.0079) but a significantly higher left ventricular ejection fraction (63+/-10% versus 52+/-10%, P=0.0009) despite a similar degree of diameter stenosis (67+/-13% versus 68+/-16%, P=NS). A significant inverse correlation was found between LVEF and FFR (R=0.29, P=0.049). CONCLUSIONS: The present data indicate (1) that the 0.75 cutoff value of FFR to distinguish patients with positive from patients with negative SPECT imaging is valid after a myocardial infarction and (2) that for a similar degree of stenosis, the value of FFR depends on the mass of viable myocardium.

Membrane lymphotoxin is required for the development of different subpopulations of NK T cells.

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTalpha and LTbeta, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTalpha-/- and LTbeta-/- mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTalpha-/- and LTbeta-/- mice did not respond to the lipoglycan alpha-galactosylceramide, which is presented by mouse CD1 to Valpha14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and alpha-galactosylceramide reactive and those that are not, were affected by disruption of the LTalpha and LTbeta genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTbeta receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Estimation of risk based on biological dosimetry for patients treated with radioiodine.

A multicentre study was undertaken to assess the cytogenetic damage to peripheral blood lymphocytes in 31 patients treated with 131I for thyrotoxicosis using the cytokinesis-blocked micronucleus assay. The results were compared to those for eight thyroid carcinoma patients using the same method. For each patient, blood samples were taken immediately before and 1 week after iodine administration. The first blood sample was divided into three fractions and each fraction was subsequently irradiated in vitro with 0, 0.5 and 1 Gy 60Co gamma rays, respectively. After blood culture for 70 h, cells were harvested, stained with Romanowsky-Giemsa and the micronuclei scored in 1000 binucleated cells. For both patient groups, a linear-quadratic dose-response curve was fitted through the data set of the first blood sample by a least squares analysis. The mean increase in micronuclei after 131I therapy (second blood sample) was fitted to this curve and the mean equivalent total body dose (ETBD) calculated. Surprisingly, in view of the large difference in administered activity between thyroid carcinoma patients and thyrotoxicosis patients, the increase in micronuclei after therapy (mean +/- S.D.: 32 +/- 30 and 32 +/- 23, respectively) and the equivalent total body dose (0.34 and 0.32 Gy, respectively) were not significantly different (P > 0.1). The small number of micronuclei induced by 131I therapy (32 +/- 29), compared with external beam radiotherapy for Hodgkin's disease (640 +/- 381) or cervix carcinoma (298 +/- 76) [1], gave a cancer mortality estimate of less than 1%. This also explains why late detrimental effects in patients after 131I treatment have not been reported in the literature.

Mucosal immunity and inflammation. II. The yin and yang of T cells in intestinal inflammation: pathogenic and protective roles in a mouse colitis model.

Inflammatory bowel disease (IBD) is a multifactorial immune disorder of uncertain etiology. The advent of several mouse models of mucosal inflammation that resemble IBD has provided insight into the mechanisms governing both normal and pathological mucosal immune function. In a widely used adoptive transfer model, the injection into immunodeficient mice of a subset of CD4(+) T lymphocytes, the CD4(+)CD45RBhigh cells, leads to inflammation of the intestine. Pathogenesis is due in part to the secretion of proinflammatory cytokines. The induction of colitis can be prevented by cotransfer of another CD4(+) subpopulation, the CD4(+)CD45RBlow T cells. This population behaves analogously to the CD4(+)CD45RBhigh population in terms of the acquisition of activation markers and homing to the host intestine. However, their lymphokine profile when activated is different, and anti-inflammatory cytokines secreted and/or induced by CD4(+)CD45RBlow T cells prevent colitis. In this themes article, a description of the adoptive transfer model is given, the factors that promote and prevent colitis pathogenesis are discussed, and some controversial aspects of the model are addressed.

Real-life radiation burden to relatives of patients treated with iodine-131: a study in eight centres in Flanders (Belgium).

In view of the EURATOM 96/29 [1] regulations, a prospective multicentre study was performed to evaluate the present guidelines given to relatives of patients treated with iodine-131 for both thyroid carcinoma and thyrotoxicosis, based on the real-life radiation burden. This study comprised 166 measurements carried out on a group of 94 relatives of 65 patients. All relatives wore a thermoluminescent dosemeter (TLD) on the wrist for 7 days. Sixty-one relatives agreed to wear another TLD for an additional 7 days. TLD were placed on nine patients' bedside tables. The eight participating centres were arbitrarily divided into three groups according to the period of time they advised their patients to sleep separately. Groups I, II and III respectively advised their patients to sleep separately for 0, 7-10 and 14-21 days. The median dose received by in-living relatives of thyroid carcinoma patients during the 14 days following hospital discharge was 281 microSv (doses to infinity not calculated); the median dose to infinity received by in-living relatives of ambulatory treated thyrotoxicosis patients was 596 microSv, as compared with 802 microSv for in-living relatives of hospitalised thyrotoxicosis patients. In general the children of patients received a significantly (P < 0.1) lower mean dose than their partners. For thyroid carcinoma patients, only two relatives out of 19 (10%) exceeded the EURATOM 96/29 limit of 1 mSv/year. For thyrotoxic patients, 28% of relatives exceeded the EURATOM 96/29 limit, but none of them were relatives of patients who followed guidelines for 21 days. The results of this study indicate that sleeping separately for 7 days, after a period of hospitalisation of 2-3 days, will usually be sufficient for thyroid carcinoma patients. For thyrotoxicosis patients, up to 21 days of sleeping separately could be necessary in order to strictly abide by EURATOM 96/29. Therefore, the authors propose the implementation of a non-rigid dose constraint for people who "knowingly and willingly" help patients treated with 131I, while still following the ALARA principle.

Value of axillary ultrasonography and sonographically guided puncture of axillary nodes: a prospective study in 144 consecutive patients.

The value of axillary sonography in 144 consecutive patients was prospectively studied. Abnormal lymph nodes were demonstrated in 72 axillae, only half (36 of 72) of which were clinically detected. In comparison with intraoperative findings in 47 patients with breast carcinoma, the sensitivity of sonography in detecting malignant nodes was 92%, the specificity 95%, and the positive and negative predictive value 96% and 91%, respectively. In 42 patients a sonographically guided node puncture was performed. Evidence of malignancy was found in 38 (90.5%). In 14 (33%) of them, cyto-histology pointed to a clinically undetected primary malignancy.

alpha-Amino acids derived from ornithine as building blocks for peptide synthesis.

Recently great interest has arisen in the synthesis of combinatorial libraries, and this technology provides a significant partner to contemporary strategies in rational design and lead discovery. By simple combination of a given set of building blocks, high numbers of different molecules are produced simultaneously, increasing the possibility of discovery of a lead compound in a limited time. One direction of research in this field focuses on the synthesis of libraries composed of modified amino acids. Here, the synthesis and characteristics of some building blocks derived from ornithine are described. The synthesis is based on the acylation/sulfonation of the copper complex of ornithine by aroyl and arylsulfonyl chlorides exemplified by 2-thiophenecarbonyl chloride, p-toluenesulfonyl chloride and 8-quinolinesulfonyl chloride. To evaluate the potential use of these modified alpha-amino acids as component in an oligopeptide library, all three derivatives were incorporated in a hexapeptide with a random sequence using a standard coupling procedure (DIC/HOBt/DIEA). Depending upon the acidity of the amido hydrogen on the delta-nitrogen, competition between intramolecular cyclization and peptide bond formation was observed. The higher the acidity, the more pronounced is this side reaction. Coupling conditions for peptide formation were optimized so that the newly described amino acid based building blocks are suitable for incorporation into libraries consisting of unnatural amino acids. The outlined procedures open up a broad avenue of possibilities for creation of diversity into peptidic libraries.

Understanding the binding of 5-substituted 2'-deoxyuridine substrates to thymidine kinase of herpes simplex virus type-1.

Thymidine kinase from HSV-1 (HSV-1 TK) is a key enzyme in the metabolic activation of antiviral nucleosides. High affinity of such compounds for the enzyme is required for efficient phosphorylation. In this study, affinity data from a series of 5-substituted 2'-deoxyuridine substrates in combination with the crystal structure of the viral enzyme were used to investigate the structural factors influencing the affinity of these compounds for the enzyme. Calculations showed that the binding energetics and conformations of thymidine and the 5-substituted 2'-uridine analogues are similar. The major part of the binding energy arises from interactions involving sugar and base moieties. Small differences in affinity for the enzyme are explained by the hydrophobicity of the 5-substituent or by its energetic complementarity with the active site pocket. In designing high-affinity nucleoside substrates of HSV-1 TK, care should be taken to maintain the geometry of the base moiety and sugar hydroxyl functionalities. Substitutions at the 5-position of the nucleobase should be lipophilic and characterized by well-defined geometrical properties. The present study represents a first quantitative explanation for HSV-1 TK affinity of 5-substituted 2'-deoxyuridines which are historically the first group of selective antivirals. The results may be used to design new and more potent compounds.

Aldose reductase as a target for drug design: molecular modeling calculations on the binding of acyclic sugar substrates to the enzyme.

In an attempt to obtain a picture of the binding conformation of aldehyde substrates to human aldose reductase (hAR), modeling calculations have been performed on the binding of three substrates, D-xylose, L-xylose, and D-lyxose, to wild-type human aldose reductase and two of its site-directed mutants. It was found that the average geometry of D-xylose in the active site of wild-type aldose reductase is characterized by strong hydrogen bonds involving the reactive carbonyl oxygen of the substrate and both Tyr48 and His110. The calculations also suggest the importance of Trp111 in the binding of 2'-hydroxyl-containing aldehyde substrates. A good correlation between calculated interaction enthalpies and experimental log(Km) or log(kcat/Km) values was obtained when His110 was modeled with its N epsilon 2 atom protonated and N delta 1 unprotonated. No correlation was found for the other two configurations of His110. On the basis of comparisons of the calculated substrate binding conformations for the three possible His110 configurations, and on the correlations between measured log(Km) or log(kcat/Km) and calculated parameters, it is proposed that His110 is neutral and protonated at N epsilon 2 when an aldehyde substrate is bound to the hAR/NADPH complex. A chain of three hydrogen-bonded water molecules has been identified in all available crystal structures and is located in an enzyme channel which links the N delta 1 atom of His110 to the solvent-accessible surface of the enzyme. A possible role of this channel in the mechanism of catalysis of aldose reductase is suggested.

1-(2,3-Dideoxy-erythro-beta-D-hexopyranosyl)cytosine: an example of the conformational and stacking properties of pyranosyl pyrimidine nucleosides. A crystallographic and computational approach.

C10H15N3O4, Mr = 241.25, orthorhombic, P2(1)2(1)2(1), a = 7.4013 (4), b = 8.7563 (5), c = 17.392 (1) A, V = 1127.1 (1) A3, Z = 4, Dm = 1.42, Dx = 1.422 Mg m-3, Ni-filtered Cu K alpha radiation, lambda = 1.54178 A, mu = 0.895 mm-1, F(000) = 512, T = 293 K, final R = 0.044 for 1024 unique observed [F greater than or equal to 6 sigma (F)] reflections. The conformational parameters are in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature [Pure Appl. Chem. (1983), 55, 1273-1280] guidelines. In order to assess the possible use of pyranosyl-modified pyrimidine nucleosides in the design of new synthetic oligonucleotides, the conformational and packing properties of 13 structures were examined. From this study, it becomes clear that the pyrimidine-base geometry is independent of the sugar ring type (furanosyl- or pyranosyl-like). The bases are always positioned in an equatorial orientation on the pyranoside sugar, which means that the sugar adopts a 4C1 conformation in alpha- and 4C1 in beta-enantiomers. As a result of the anomeric effect the O5'-C1' bond length is 0.020 (4) A shorter than the C5'-O5' distance (C1' is the anomeric C atom). The O5'-C1'-N1-C2 torsion angle chi in the 13 nucleosides is centered around 244 (8) degrees and varies from 196.4 (3) to 287.0 (2) degrees. Molecular-mechanics calculations on uncharged pyranosyl nucleosides are found to be less accurate compared with semi-empirical quantum-chemical methods or molecular-mechanics calculations on charged molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass concentration measurements of creatine kinase BB isoenzyme as an index of brain tissue damage.

A new two-site immunoenzymometric method using monoclonal antibodies was developed for measuring CK-BB mass concentrations in cerebrospinal fluid (CSF). Within- and between-assay coefficient of variation values for the method varied between 6 and 9%. Assay results are not affected by presence of sulfate and sialic acid groups on the enzyme. In comparison to catalytic activity measurements, a steady decline in the enzyme's specific activity was observed after acute head trauma. Repetitive measurements of CK-BB mass concentration in cerebrospinal fluid during the first 24 h after trauma enabled the estimation of brain lesion size. Clinical outcome of acute head trauma patients evaluated by Glasglow Outcome Scale, correlated well with cumulative CK-BB release after trauma. Also in neonates, CK-BB determinations in CSF correlated well with clinical findings.

Structure of the neuroleptic drug 4-amino-N-1-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(ethylsulfonyl)-2- methoxybenzamide (amisulpride).

C17H27N3O4S, Mr = 369.48, monoclinic, P2(1)/c, a = 13.333 (7), b = 7.946 (4), c = 17.550 (10) A, beta = 96.99 (4) degrees, V = 1845 (2) A3, Z = 4, Dm = 1.33, Dx = 1.330 Mg m-3, graphite-monochromated Cu K alpha radiation, lambda = 1.54178 A, mu = 1.744 mm-1, F(000) = 792, T = 293 K. Final R = 0.038 for 2405 unique observed reflections. The folded conformation of the molecule with the least-squares planes of the aromatic and the pyrrolidine rings almost perpendicular is essentially determined by intra- and intermolecular hydrogen bonds. In this way, two pseudorings are formed, one linking the amide H with the methoxy O, and a second one involving the 4-amino H and a sulfonyl O. An intermolecular hydrogen bond forces the planar amide group some 28 degrees out of the plane of the aromatic ring.