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Essentials N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous (SC) FVIII dosing might reduce the treatment burden of prophylaxis. SC N8-GP has a favorable PK profile in animal models and disappears from skin injection sites. Combined animal (SC) and clinical (IV) data suggest that daily SC dosing may provide prophylaxis., Summary: Background N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals. Objective To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8-GP in preclinical models and predict the human pharmacokinetic (PK) profile. Methods The pharmacokinetics of subcutaneously administered N8-GP were evaluated in FVIII knockout (F8-KO) mice and cynomolgus monkeys; a human PK prediction model in hemophilia A patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8-KO mice. The injection-site distribution and absorption of subcutaneously administered N8-GP were assessed in F8-KO mice by the use of temporal fluorescence imaging and immunohistochemistry. Results Subcutaneously administered N8-GP had a bioavailability, a first-order absorption rate and a half-life, respectively, of 24%, 0.094 h -1 and 14 h in F8-KO mice, and 26%, 0.33 h -1 and 15 h in cynomolgus monkeys. A dose-dependent effect of subcutaneously administered N8-GP on blood loss was observed in mice. A minimal amount of N8-GP was detected at the injection site 48-72 h after single or multiple dose(s) in F8-KO mice. Subcutaneously administered N8-GP was localized to the skin around the injection site, with time-dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8-GP at a daily dose of 12.5 IU kg -1 will provide FVIII trough levels of 2.5-10% in 95% of patients with severe hemophilia A. Conclusions Subcutaneously administered N8-GP may provide effective hemophilia A prophylaxis. A phase I clinical trial is underway to investigate this possibility., (© 2018 Novo Nordisk A/S. Journal of Thombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society of Thombosis and Haemostasis.)

Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by glycogen branching enzyme (GBE) deficiency and resulting in the storage of abnormal glycogen (polyglucosan). Prenatal diagnosis is based on biochemical assay of GBE activity or on mutation analysis, but polyglucosan can also be identified histologically in fetal tissues. We document placental involvement at 25 and 35 weeks of gestation in two cases with genetically confirmed GSD IV. Intracellular inclusions were seen mainly in the extravillous trophoblast. Our findings suggest the possibility of prenatal diagnosis by histological evaluation of placental biopsies.

Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.

Efficient gene control by antisense RNA requires rapid bi-molecular interaction with a cognate target RNA. A comparative analysis revealed that a YUNR motif (Y=pyrimidine, R=purine) is ubiquitous in RNA recognition loops in antisense RNA-regulated gene systems. The (Y)UNR sequence motif specifies two intraloop hydrogen bonds forming U-turn structures in many anticodon-loops and all T-loops of tRNAs, the hammerhead ribozyme and in other conserved RNA loops. This structure creates a sharp bend in the RNA phosphate-backbone and presents the following three to four bases in a solvent-exposed, stacked configuration providing a scaffold for rapid interaction with complementary RNA. Sok antisense RNA from plasmid R1 inhibits translation of the hok mRNA by preventing ribosome entry at the mok Shine & Dalgarno element. The 5' single-stranded region of Sok-RNA recognizes a loop in the hok mRNA. We show here, that the initial pairing between Sok antisense RNA and its target in hok mRNA occurs with an observed second-order rate-constant of 2 x 10(6) M(-1) s(-1). Mutations that eliminate the YUNR motif in the target loop of hok mRNA resulted in reduced antisense RNA pairing kinetics, whereas mutations maintaining the YUNR motif were silent. In addition, RNA phosphate-backbone accessibility probing by ethylnitrosourea was consistent with a U-turn structure formation promoted by the YUNR motif. Since the YUNR U-turn motif is present in the recognition units of many antisense/target pairs, the motif is likely to be a generally employed enhancer of RNA pairing rates. This suggestion is consistent with the re-interpretation of the mutational analyses of several antisense control systems including RNAI/RNAII of ColE1, CopA/CopT of R1 and RNA-IN/RNA-OUT of IS10., (Copyright 1999 Academic Press.)

Wrist arthroscopy has become an accepted diagnostic technique, and it is starting to be a useful therapeutic tool. Extensor tendons, the radial artery, and dorsal sensory nerve branches are at risk of injury during this procedure; however, understanding periportal anatomy should make wrist arthroscopy safer. Wrist arthroscopic portals were established in 19 fresh cadaver wrists, after which the limbs were dissected and periportal anatomy was described and quantified. The 1-2, 6R, and 6U portals were the most perilous, while the midcarpal, 3-4, 4-5, and distal radioulnar joint portals were relatively safe. Even "safe" portals had occasional adjacent sensory nerve branches and tendons. A safe technique of establishing wrist arthroscopy portals is emphasized.

Administration of vecuronium by infusion is an increasingly common technique, both in the operating room and in the intensive care unit (ICU), for patients requiring prolonged neuromuscular blockade and mechanical ventilation. The major advantage of vecuronium over older neuromuscular blocking agents is its rapid excretion and intermediate duration of action. Prior to the current case report, the longest reported continuous paralysis after the cessation of a vecuronium infusion was 90 hours. A case of an 81-year-old patient with renal failure and subclinical chronic cirrhosis of the liver, who remained paralyzed for 13 days following a vecuronium infusion, is described. Intensive monitoring of neuromuscular function is recommended whenever muscle relaxants are administered by continuous infusion.

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15-405 micrograms/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 micrograms/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.

We have investigated a relationship between two detoxication systems, metabolic detoxication through the cytochrome P-450 (P-450) pathway and resistance to infection through interferon (IFN), in mice infected with influenza virus following exposure to coal dust (CD) and diesel exhaust (DE) particulates. Mice were exposed by inhalation to filtered air (FA; control), CD, or DE for 1 month and then inoculated intranasally (IN) with influenza virus. During infection, 7-ethoxycoumarin deethylase (7ECdeEt'ase) and ethylmorphine demethylase (EMdeMe'ase) (monooxygenases), and NADPH cytochrome c reductase (NADPH c red'ase) were measured in liver microsomes. Temporal patterns of enzyme activities were observed with control animals. EMdeMe'ase and NADPH c red'ase exhibited peak values at Day 4 postinfection (27.6 and 482 nmole/min/mg protein, respectively), compared to initial activities (9.1 and 307 nmole/min/mg protein, respectively). 7ECdeEt'ase activity decreased between Days 1-3 postvirus infection and thereafter returned to the original value (1.7 nmole/min/mg protein). When the mice were first exposed to CD or DE particulates for 1 month prior to influenza infection, changes in enzyme temporal patterns were observed. The increased EMdeMe'ase activity at Day 4 was not observed in mice exposed to CD and was reduced in mice exposed to DE. Preexposure to either particulate resulted in the abolition of the increased Day 4 activity of NADPH c red'ase. The 7ECdeEt'ase postinfection temporal pattern was not affected by a preexposure to either particulate. Estimates of the enzyme activities after the 1-month exposure to FA, CD, or DE but before virus infection indicated no changes due to particulate exposure alone. Under these conditions of particulate exposure and virus infection, serum IFN levels in the mice used in this study peaked at Days 4-5 and were unaffected by the 1-month preexposure to CD or DE (Hahon et al., (1985). The data suggest the relationship that exists between metabolic detoxication and resistance to infection in normal mice was altered during a short-term preexposure to CD or DE.

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.

As an extension of earlier work on aliphatic alpha-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic alpha-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilylation. The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0 degrees. The final derivatives are stable for weeks at room temperature. Low resolution mass spectra are reported. The fragmentation mechanisms are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinois, O-(TMS-d9)-quinoxalinois and O-TMS-6(7)-chloroquinoxalinois.

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.