A study of the reactivity of S(VI)-F containing warheads with nucleophilic amino-acid side chains under physiological conditions.

Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compounds, a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related SVI-F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aqueous solutions, indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-containing covalent probe compounds and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.

Putting patients at the center of kidney care transitions: PREPARE NOW, a cluster randomized controlled trial.

Care for patients transitioning from chronic kidney disease to kidney failure often falls short of meeting patients' needs. The PREPARE NOW study is a cluster randomized controlled trial studying the effectiveness of a pragmatic health system intervention, 'Patient Centered Kidney Transition Care,' a multi-component health system intervention designed to improve patients' preparation for kidney failure treatment. Patient-Centered Kidney Transition Care provides a suite of new electronic health information tools (including a disease registry and risk prediction tools) to help providers recognize patients in need of Kidney Transitions Care and focus their attention on patients' values and treatment preferences. Patient-Centered Kidney Transition Care also adds a 'Kidney Transitions Specialist' to the nephrology health care team to facilitate patients' self-management empowerment, shared-decision making, psychosocial support, care navigation, and health care team communication. The PREPARE NOW study is conducted among eight [8] outpatient nephrology clinics at Geisinger, a large integrated health system in rural Pennsylvania. Four randomly selected nephrology clinics employ the Patient Centered Kidney Transitions Care intervention while four clinics employ usual nephrology care. To assess intervention effectiveness, patient reported, biomedical, and health system outcomes are collected annually over a period of 36 months via telephone questionnaires and electronic health records. The PREPARE NOW Study may provide needed evidence on the effectiveness of patient-centered health system interventions to improve nephrology patients' experiences, capabilities, and clinical outcomes, and it will guide the implementation of similar interventions elsewhere. TRIAL REGISTRATION: NCT02722382.

Structural Characterisation Reveals Mechanism of IL-13-Neutralising Monoclonal Antibody Tralokinumab as Inhibition of Binding to IL-13Rα1 and IL-13Rα2.

Interleukin (IL)-13 is a pleiotropic T helper type 2 cytokine frequently associated with asthma and atopic dermatitis. IL-13-mediated signalling is initiated by binding to IL-13Rα1, which then recruits IL-4Rα to form a heterodimeric receptor complex. IL-13 also binds to IL-13Rα2, considered as either a decoy or a key mediator of fibrosis. IL-13-neutralising antibodies act by preventing IL-13 binding to IL-13Rα1, IL-4Rα and/or IL-13Rα2. Tralokinumab (CAT-354) is an IL-13-neutralising human IgG4 monoclonal antibody that has shown clinical benefit in patients with asthma. To decipher how tralokinumab inhibits the effects of IL-13, we determined the structure of tralokinumab Fab in complex with human IL-13 to 2 Å resolution. The structure analysis reveals that tralokinumab prevents IL-13 from binding to both IL-13Rα1 and IL-13Rα2. This is supported by biochemical ligand-receptor interaction assay data. The tralokinumab epitope is mainly composed of residues in helices D and A of IL-13. It is mostly light chain complementarity-determining regions that are driving paratope interactions; the variable light complementarity-determining region 2 plays a key role by providing residue contacts for a network of hydrogen bonds and a salt bridge in the core of binding. The key residues within the paratope contributing to binding were identified as Asp50, Asp51, Ser30 and Lys31. This study demonstrates that tralokinumab prevents the IL-13 pharmacodynamic effect by binding to IL-13 helices A and D, thus preventing IL-13 from interacting with IL-13Rα1 and IL-13Rα2.

Failure of the antioxidant vitamin E to protect against adriamycin-induced cardiotoxicity in the rabbit.

Recently, vitamin E has been proposed to protect against Adriamycin-induced cardiotoxicity. We studied contractile decline and ultramicroscopic alterations of the heart of rabbits chronically treated with Adriamycin up to a cumulative dose of 400 mg/sq m. High doses of vitamin E did not protect against the Adriamycin-induced development of severe contractile decline as evaluated by means of measurement of the interval-force relationship curve. Light and electron microscopic analysis did not show any signs of protection against Adriamycin-induced morphological alterations. Biochemical and hematological alterations. Biochemical and hematological alterations caused by the antineoplastic agent were similar in both Adriamycin-treated animal groups. Coadministration of vitamin E did result in an increased life span. This study indicates that vitamin E does not protect against the development of cardiomyopathy and contractile decline after chronic exposure to Adriamycin.

Polarity-dependent conformational switching of a peptide mimicking the S4-S5 linker of the voltage-sensitive sodium channel.

The S4-S5 linker (or S45) in voltage-sensitive sodium channels was previously shown to be involved in the permeation pathway. The secondary structure, investigated by circular dichroism, of a S4-S45 peptide from domain IV and its fragments (including S45) is reported here and compared with that of the homologous peptide from domain II as a function of the solvent dielectric constant. The reduction in helicity seen for S4-S45 (II) in polar media is cancelled in membrane-like environment. The most striking result-- a sharp alpha-helix --> beta-sheet transition upon exposure of the S45 moiety to aqueous solvents-- is discussed as regards channel activation and selectivity.

Ion channel stability and hydrogen bonding. Molecular modelling of channels formed by synthetic alamethicin analogues.

Several analogues of the channel-forming peptaibol alamethicin have been demonstrated to exhibit faster switching between channel substates than does unmodified alamethicin. Molecular modelling studies are used to explore the possible molecular basis of these differences. Models of channels formed by alamethicin analogues were generated by restrained molecular dynamics in vacuo and refined by short molecular dynamics simulations with water molecules within and at either mouth of the channel. A decrease in backbone solvation was found to correlate with a decrease in open channel stability between alamethicin and an analogue in which all alpha-amino-isobutyric acid residues of alamethicin were replaced by leucine. A decrease in the extent of hydrogen-bonding at residue 7 correlates with lower open channel stabilities of analogues in which the glutamine at position 7 was replaced by smaller polar sidechains. These two observations indicate the importance of alamethicin/water H-bonds in stabilizing the open channel.

Alamethicin channels - modelling via restrained molecular dynamics simulations.

Alamethicin channels have been modelled as approximately parallel bundles of transbilayer helices containing between N = 4 and 8 helices per bundle. Initial models were generated by in vacuo restrained molecular dynamics (MD) simulations, and were refined by 60 ps MD simulations with water molecules present within and at the mouths of the central pore. The helix bundles were stabilized by networks of H-bonds between intra-pore water molecules and Gln-7 side-chains. Channel conductances were predicted on the basis of pore radius profiles, and suggested that the N = 4 bundle formed an occluded pore, whereas pores with N > or = 5 helices per bundle were open. Continuum electrostatics calculations suggested that the N = 6 pore is cation-selective, whereas pores with N > or = 7 helices per bundle were predicted to be somewhat less ion-selective.

Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and long-term safety data.

PURPOSE: Tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) is inhibited by the recombinant humanized (rhu) monoclonal antibody (MAb) rhuMAbVEGF, which has synergy with chemotherapy in animal models. The present study was designed to assess the safety and pharmacokinetics of weekly intravenous (IV) rhuMAbVEGF with one of three standard chemotherapy regimens. PATIENTS AND METHODS: Twelve adult patients were enrolled four on each combination. rhuMAbVEGF, 3 mg/kg IV, was administered weekly for 8 weeks with (1) doxorubicin 50 mg/m(2) every 4 weeks; (2) carboplatin at area under the curve of 6 plus paclitaxel 175 mg/m(2) every 4 weeks; and (3) fluorouracil (5-FU) 500 mg/m(2) with leucovorin 20 mg/m(2) weekly, weeks 1 to 6 every 8 weeks. RESULTS: The median number of rhuMAbVEGF doses delivered was eight (range, four to eight doses). Grade 3 toxicities were diarrhea (one 5-FU patient), thrombocytopenia (two patients on carboplatin plus paclitaxel), and leukopenia (one patient on carboplatin plus paclitaxel). These toxicities were likely attributable to the chemotherapy component of the regimen. The mean (+/- SD) peak serum level of rhuMAbVEGF was 167 +/- 46 microg/mL, and the mean terminal half-life was 13 days. Total (free plus bound) serum VEGF levels increased from 51 +/- 39 pg/mL (day 0) to 211 +/- 112 (day 49) pg/mL. Three responding patients continued treatment with rhuMAbVEGF and chemotherapy, receiving the equivalent of 36, 20, and 40 total rhuMAbVEGF doses with no cumulative or late toxicities. CONCLUSION: rhuMAbVEGF can be safely combined with chemotherapy at doses associated with VEGF blockade and without apparent synergistic toxicity. Its contribution to the treatment of advanced solid tumors should be evaluated in randomized treatment trials.

Prediction by a neural network of outer membrane beta-strand protein topology.

An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) beta-strand proteins. Specifically, the NN predicts the z-coordinate of Calpha atoms in a coordinate frame with the outer membrane in the xy-plane, such that low z-values indicate periplasmic turns, medium z-values indicate transmembrane beta-strands, and high z-values indicate extracellular loops. To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z-axis. The relationship between Calpha z-values and topology was thereby established. To predict the topology of other OM proteins, all seven porins were used for the training set. Z-values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set. The results of topology prediction compare favorably with experimental topology data.

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Regional specificity of testosterone regulation of proopiomelanocortin gene expression in the arcuate nucleus of the male rat brain.

Endogenous opioid peptides have been implicated as mediators in the negative feedback action of gonadal steroids on GnRH secretion. We have previously demonstrated that testosterone stimulates POMC gene expression in neurons of the arcuate nucleus. However, the wide distribution and variety of actions attributed to the numerous arcuate POMC neurons suggest that these cells may be heterogeneous in their responsiveness to steroid hormones. We tested the hypothesis that testosterone modulates a select population of POMC neurons within the arcuate nucleus of the adult male rat by comparing POMC mRNA signal levels throughout the arcuate nucleus of intact, castrated, and castrated testosterone-replaced adult males. Adult male rats were castrated and implanted (sc) with a Silastic capsule (30 mm) that was either empty (n = 6) or filled with crystalline testosterone (n = 5). Control sham-operated animals (n = 6) were left intact. In each animal the arcuate nucleus was divided into four equal rostral-caudal areas within which we measured POMC mRNA content in individual cells. We report that the effects of castration and testosterone replacement are observed in POMC neurons located in the most rostral region of the arcuate nucleus. After castration, POMC mRNA content was reduced in cells of the most rostral arcuate area (intact, 152 +/- 5 grains/cell; castrate, 119 +/- 2 grains/cell; P less than 0.0005), and replacement with physiological levels of testosterone prevented the decline in POMC mRNA levels so that they remained equivalent to that of the intact animal (castrated testosterone-replaced, 153 +/- 6 grains/cell). There was no significant difference in POMC mRNA signal between intact and castrated testosterone-replaced animals in the most rostral area. POMC neurons in the more caudal aspect of the arcuate (75% of the nucleus) were unaffected by the treatments; alternatively, it is possible that a real change in POMC message content in a subpopulation of cells was obscured by larger numbers of nonresponding cells within the same tissue sections. Based on these observations we conclude that there is a heterogeneous population of POMC neurons in the arcuate nucleus and that testosterone regulates POMC gene expression in a select group of these cells located in the rostral portion of the arcuate nucleus.

Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain.

Gender differences in hypothalamic somatostatin (SS) secretion may account in part for the sexually dimorphic patterns of GH secretion in rats. Since males have lower baseline serum GH levels than females, and SS inhibits GH secretion, we hypothesized that the SS neurons in the periventricular nucleus (PeN) of the male rat would have greater biosynthetic activity than those of the female. We tested this hypothesis by measuring SS mRNA in cells in the PeN of intact male and proestrous female rats. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA content in individual cells in the PeN and compared signal levels (autoradiographic grains per cell) between male and proestrous female animals. The signal level of SS mRNA in cells of the PeN was significantly greater in males than in proestrous females (males, 210 +/- 7 grains/cell; females, 158 +/- 5 grains/cell; P less than 0.0005), whereas no difference was observed in SS cells of the frontal cortex (males, 100 +/- 0.8 grains/cell; females, 99 +/- 5.9 grains/cell). This difference in SS mRNA levels is likely to be the result of different hormonal environments exerting an influence on neurons of the hypothalamus. To test the hypothesis that testosterone stimulates SS gene expression in neurons of the PeN, adult male rats were castrated and immediately implanted with either empty (sham; n = 3) or testosterone-containing (n = 3) Silastic implants of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that castrated animals had significantly lower levels of SS mRNA signal in neurons of the PeN compared with intact animals (intact, 195 +/- 3 grains/cell; castrated, 159 +/- 6 grains/cell; P less than 0.003) and that physiological levels of testosterone prevent this reduction in SS mRNA levels (castrated testosterone-replaced, 182 +/- 4 grains/cell; castrated, 159 +/- 6 grains/cell; P less than 0.003). Furthermore, testosterone-treated castrates had SS mRNA signal levels indistinguishable from those of intact controls (intact, 195 +/- 3 grains/cell; castrated testosterone-replaced, 182 +/- 4 grains/cell). There was no significant difference in SS mRNA levels in neurons of the frontal cortex (intact, 98 +/- 2 grains/cell; castrated, 98 +/- 3 grains/cell; castrated testosterone-replaced, 102 +/- 2 grains/cell).(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone-releasing hormone messenger ribonucleic acid in the hypothalamus of the adult male rat is increased by testosterone.

Since intact adult male rats have higher GH pulse amplitude than do castrated animals and since GH-releasing hormone (GHRH) secretion is predominantly responsible for the production of these GH pulses, we hypothesized that testosterone stimulates GHRH synthesis in neurons of the hypothalamus. To test this hypothesis, we compared GHRH mRNA content in individual neurons of the arcuate (ARC) and ventromedial (VMH) nuclei among groups of intact (n = 3), castrated (n = 5), and castrated testosterone-replaced (n = 5) adult male rats. Cellular GHRH mRNA content was measured by using semiquantitative in situ hybridization with an 35S-labeled cRNA probe complementary to the coding sequence of rat GHRH mRNA. Castration resulted in an approximately 35% decline in GHRH mRNA signal relative to that in intact animals in both the ARC (P less than 0.005) and VMH (P less than 0.005). Replacement with testosterone at the time of castration completely prevented the decline in both areas. Testosterone can exert effects either through activation of the androgen receptor directly or through aromatization to estradiol; therefore, we also examined the effects on GHRH mRNA of replacement with 17 beta-estradiol (n = 5) or dihydrotestosterone (DHT), a nonaromatizable androgen (n = 4). Estradiol had no effect on the castration-induced decline in GHRH mRNA in either the ARC or VMH. In contrast, DHT partially prevented the postcastration decline in GHRH in the ARC (P less than 0.005), while having no statistically significant effect on GHRH mRNA in the VMH. These results clearly indicate that testosterone stimulates expression of GHRH mRNA in neurons of the hypothalamus. Furthermore, the failure of estradiol to substitute for testosterone and the ability of DHT to substantially support GHRH mRNA suggest that testosterone exerts its effects on GHRH gene expression predominantly through direct activation of the androgen receptor.

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat.

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.

Conservation analysis and structure prediction of the SH2 family of phosphotyrosine binding domains.

Src homology 2 (SH2) regions are short (approximately 100 amino acids), non-catalytic domains conserved among a wide variety of proteins involved in cytoplasmic signaling induced by growth factors. It is thought that SH2 domains play an important role in the intracellular response to growth factor stimulation by binding to phosphotyrosine containing proteins. In this paper we apply the techniques of multiple sequence alignment, secondary structure prediction and conservation analysis to 67 SH2 domain amino acid sequences. This combined approach predicts seven core secondary structure regions with the pattern beta-alpha-beta-beta-beta-beta-alpha, identifies those residues most likely to be buried in the hydrophobic core of the native SH2 domain, and highlights patterns of conservation indicative of secondary structural elements. Residues likely to be involved in phosphotyrosine binding are shown and orientations of the predicted secondary structures suggested which could enable such residues to cooperate in phosphate binding. We propose a consensus pattern that encapsulates the principal conserved features of the SH2 domains. Comparison of the proposed SH2 domain of akt to this pattern shows only 12/40 matches, suggesting that this domain may not exhibit SH2-like properties.

The structure of porin from Paracoccus denitrificans at 3.1 A resolution.

The crystal structure of a non-specific porin from Paracoccus denitrificans at 3.1 A resolution has been solved by molecular replacement using the porin from Rhodopseudomonas blastica as the search model. Paracoccus porin is very similar to other non-specific porins of known structure: a trimer of 16 stranded beta-barrels each with a central pore constricted by a long extracellular loop folding back against the barrel wall. The distinctive distribution of charged residues of this non-specific porin contributes to understanding the relation between structure and ion selectivity.

The influence of perindopril and the diuretic combination amiloride+hydrochlorothiazide on the vessel wall properties of large arteries in hypertensive patients.

OBJECTIVE: To compare the cardiovascular effects of 6 months of treatment with the angiotensin converting enzyme inhibitor perindopril and with the diuretic combination amiloride+hydrochlorothiazide, and to study possible persistence of observed treatment effects after discontinuation of antihypertensive therapy. DESIGN: A placebo run-in period preceded a 6-month active-treatment phase in 41 patients with essential hypertension, according to a double-blind, randomized, parallel-group design. Patients received either 4 mg perindopril or 2.5/25 mg amiloride+hydrochlorothiazide once a day. Patients were then studied for a 3-month single-blind placebo run-out period. RESULTS: After 6 months of treatment, systolic blood pressure was reduced significantly by perindopril (supine by 11%, sitting by 10%) and by amiloride+hydrochlorothiazide (supine by 8%, sitting by 12%). Diastolic blood pressure was also decreased significantly by perindopril (supine by 8%, sitting by 11%) and by amiloride+hydrochlorothiazide (supine by 4%, sitting by 9%). Mean arterial pressure decreased significantly during treatment with perindopril (by 9%) and with amiloride+hydrochlorothiazide (by 6%). Cardiac index increased with perindopril (by 6%), because of an increased stroke index (by 5%), but amiloride+hydrochlorothiazide did not change cardiac function. Systemic vascular resistance index decreased significantly more with perindopril (by 14%) than with amiloride+hydrochlorothiazide (by 8%). The distensibility of the common carotid artery was significantly enhanced by perindopril (by 16%), but not changed by amiloride+hydrochlorothiazide (1% difference). The difference between perindopril and amiloride+hydrochlorothiazide for carotid distensibility was statistically significant. The compliance of the common carotid artery tended to be increased more by perindopril (by 7%) than by amiloride+hydrochlorothiazide, which induced a 5% decrease in carotid compliance. After withdrawal of therapy, for both drugs, all treatment-induced changes were reversed to pretreatment values within 7 weeks. CONCLUSION: The distensibility of the elastic common carotid artery was increased by perindopril, but not by amiloride+hydrochlorothiazide. Large-artery properties of the muscular arteries and systemic vascular resistance improved with both drugs, but in general the changes were more pronounced with perindopril than with amiloride+hydrochlorothiazide. The present results indicate a more pronounced effect of perindopril at both macro- and microcirculatory levels, which will consequently lead to a larger decrease in cardiac afterload. After discontinuation of therapy all parameters returned to baseline values within 7 weeks.

Pharmacokinetics of a slower clearing tissue plasminogen activator variant, TNK-tPA, in patients with acute myocardial infarction.

The rapid clearance of t-PA from plasma requires administration by intravenous (I.V.) infusion. A slower clearing, fibrin-specific rt-PA variant may allow single intravenous bolus administration, thereby simplifying dosing. This study was designed to characterize the pharmacokinetics of the slower clearing, fibrin-specific tissue-plasminogen activator variant, TNK-tPA, in patients with acute myocardial infarction (AMI) following a single I.V. bolus injection. Single I.V. bolus doses of 5 to 50 mg of TNK-tPA were studied in an open-label, multicenter, dose escalation study. A total of 113 AMI patients were enrolled. Blood sampling for pharmacokinetics was conducted in eighty-two patients (72 men, 10 women), with 5 to 27 patients per dose. TNK-tPA was administered as an I.V. bolus over 5-10 s. Following I.V. bolus administration, there was a biphasic elimination of TNK-tPA from plasma. The initial phase had a mean half-life that ranged from 11 +/- 5 to 20 +/- 6 min and was followed by a terminal phase with a mean half-life that ranged from 41 +/- 16 to 138 +/- 84 min. Mean TNK-tPA plasma clearance was 125 +/- 25 - 216 +/- 98 ml/min, and the initial volume of distribution was 4.3 +/- 2 - 8.4 +/- 6 1. A decrease in TNK-tPA plasma clearance with increasing TNK-tPA dose was noted. In addition, women and patients with lower body weight or older age had a slower plasma clearance. In conclusion, TNK-tPA has a slower plasma clearance in patients with AMI than that reported for rt-PA, allowing administration as a single I.V. bolus.

A randomized trial in primary biliary cirrhosis comparing ursodeoxycholic acid in daily doses of either 10 mg/kg or 20 mg/kg. Dutch Multicentre PBC Study Group.

BACKGROUND: Ursodeoxycholic acid (UDCA) prolongs transplantation-free survival in primary biliary cirrhosis (PBC). However, the optimal therapeutic dose has not been established. AIM: To compare the effects of UDCA administered in daily doses of 10 vs. 20 mg/kg on symptoms, liver biochemistry and biliary UDCA enrichment. METHODS: A 6-month multicentre randomized open controlled trial was conducted to assess the effects of an increase in the dose of UDCA to 20 mg/kg/day vs. continuation of 10 mg/kg/day for patients who had not achieved biochemical normalization during treatment for at least 6 months with the 10 mg/kg dose. Clinical and laboratory evaluations were performed at entry and at 3-month intervals. The percentage UDCA in duodenal bile was assessed at entry and at 6 months. RESULTS: Sixty-one patients were enrolled. No side-effects of UDCA were observed. Within the 20 mg/kg/day group significant decreases were found for alkaline phosphatase (- 8%; P = 0.003), aspartate aminotransferase (- 11%; P = 0.01), alanine aminotransferase (- 17%; P < 0.001), gamma-glutamyl transferase (- 34%; P < 0.001), immunoglobulin M (- 11%; P = 0.002) and cholesterol (- 8.1%; P < 0.001). In the 10 mg/kg group none of these parameters differed significantly from baseline. No significant differences between dose groups for symptom scores or serum bilirubin were found. Biliary enrichment with UDCA increased from 37% to 46% in the 20 mg/kg group (P = 0.02) while remaining stable in the 10 mg/kg group. CONCLUSIONS: Liver biochemistry improved in PBC patients receiving UDCA 20 mg/kg/day compared to a dose of 10 mg/kg/day. Both doses were equally well tolerated. These results indicate that UDCA 10 mg/kg/ day is a suboptimal dose for treating PBC.

Dementia and the nursing home: association with care needs.

OBJECTIVE: To determine whether RUG reimbursement categories accurately predict requirements for care in nursing homes. DESIGN: Prospective descriptive study of residents in lower reimbursement categories according to RUG. SETTING: Three nursing homes in New York City. PARTICIPANTS: Convenience sample of 173 residents who agreed to participate, not significantly different from 201 who did not agree to participate. MAIN MEASURES: Chart review; assessment of residents' cognitive and functional abilities; nursing assistants' ratings of residents' functional abilities, behavioral problems, the amount of effort required in care; and time-motion studies of staff-resident interactions. RESULTS: Both the residents' RUG classification (P less than 0.01) and the level of ADL independence (P less than 0.001) had significant impacts on the staff effort required in their care, with more dependent residents requiring greater effort. The residents' level of cognitive impairment also had a significant impact on the staff effort, with the severely impaired requiring greater effort (P less than 0.05). The time-motion analysis indicated that residents within the same RUG category differed in the number of staff-resident interactions based on their level of cognitive impairment. CONCLUSIONS: Cognitive impairment is a significant morbidity (or co-morbidity) in determining the quantity of staff effort required by the resident, and behavioral interventions are an important care component. There is marked heterogeneity within lower (RUG) reimbursement categories which translates into strikingly different care requirements.