Preclinical safety and efficacy of andexanet alfa in animal models.

Essentials There is currently no approved reversal agent for factor Xa (FXa) inhibitors Andexanet alfa has been developed to reverse the anticoagulant effects of FXa inhibitors Andexanet reduced blood loss and anticoagulation markers in rivaroxaban-anticoagulated rabbits Andexanet was well tolerated in monkeys and rats, with no evidence of prothrombotic activity SUMMARY: Background Andexanet alfa is a recombinant modified form of factor Xa (FXa), designed to bind to and reverse the anticoagulant activity of FXa inhibitors. Objectives To evaluate the ability of andexanet to reverse the anticoagulant activity of rivaroxaban, and assess its pharmacokinetics (PK) and toxicity in animal models. Methods The effects of andexanet on blood loss, anti-FXa activity, rivaroxaban unbound plasma concentrations and other coagulation parameters were assessed in a rabbit liver laceration 'treatment' model. Andexanet was administered 10 min after blood loss was initiated. The toxicity of repeated administration of andexanet (up to 60 mg kg-1 day-1 ) was assessed in cynomolgus monkeys. PK parameters were evaluated in rats and monkeys. Results Excess blood loss due to anticoagulation with rivaroxaban was significantly decreased by a single intravenous bolus administration of andexanet at 35 and 75 mg per rabbit, by 75% and 63%, respectively. This correlated with dose-dependent decreases in the unbound fraction of rivaroxaban and anti-FXa activity. Co-administration of rivaroxaban had no significant impact on the PK parameters of andexanet. Andexanet (up to 60 mg kg-1 day-1 ) was well tolerated in monkeys, with no accumulation of andexanet or rivaroxaban. There was a single occurrence of anaphylaxis, which resolved after treatment with diphenhydramine and epinephrine. There was no histological evidence of prothrombotic activity with high-dose andexanet compared with vehicle control, as measured by clot and fibrin deposition in all major organs. Conclusions These data suggest that andexanet is a promising therapy for the reversal of FXa inhibitor-induced anticoagulation, supporting clinical studies in humans.

Defective gamma-glutamyl carboxylase activity and bleeding in Rambouillet sheep.

A flock of Rambouillet sheep was examined because of increased lamb mortality caused by ineffective hemostasis at parturition. Neonatal-affected lambs presented with inadequate hemostasis at the umbilicus, pale mucus membranes, and markedly prolonged activated clotting time. Affected lambs had consistently prolonged 1-stage prothrombin times and activated partial thromboplastin times that supported a defect in the common pathway or defects in both the intrinsic and extrinsic pathway of the coagulation cascade. Decreased activity of vitamin K-dependent procoagulant factors II, VII, IX, and X in male and female lambs suggested either a defect of the hepatic enzyme gamma-glutamyl carboxylase, or vitamin K(1) 2,3 epoxide reductase. Affected lamb hepatic gamma-glutamyl carboxylase activity was markedly decreased compared with that of age- and sex-matched control lambs, while vitamin K(1) 2,3 epoxide reductase and glucose-6-phosphatase activities were similar between an affected and normal lamb. Subcutaneous vitamin K(1) supplementation did not increase vitamin K-dependent procoagulant factor activities in 3 lambs administered vitamin K(1) daily. These data confirm defective gamma-glutamyl carboxylase activity as the cause of impaired coagulation of sheep in this flock. This flock represents the only viable animal model of hereditarily defective gamma-glutamyl carboxylase activity.

Truncated gamma-glutamyl carboxylase in rambouillet sheep.

A flock of Rambouillet sheep was examined because of increased lamb mortality due to ineffective hemostasis at parturition. Decreased activities of coagulation factors II, VII, IX, and X, and severely reduced hepatic gamma-glutamyl carboxylase activity with adequate vitamin K 2,3 epoxide reductase activity was determined.(1,)(21) Parenteral vitamin K(1) supplementation did not improve vitamin K-dependent coagulation factor activities in 3 affected lambs. Affected lamb gamma-glutamyl carboxylase deoxyribonucleic acid was sequenced, and 4 single nucleotide polymorphisms (SNPs 2-5) of the gamma-glutamyl carboxylase gene were identified. Single nucleotide polymorphism-4 results in an arginine to stop codon (UGA) substitution, which prematurely terminates the peptide at residue 686 (R686Stop). This genotype (GATT/GATT) has a strong association with the coagulopathy observed in clinically affected lambs, P < 0.001. The frequency of SNP-3 in exon 11 (R486H) within the MARC 1.1 database is high in the US sheep population overall. Gamma-glutamyl carboxylase activity in hepatic microsomes from a SNP-3 homozygous lamb lacking the SNP-4 mutation (GACC/GACC) was similar to control sheep homozygous for arginine at 486 and also lacking SNP-4 (TGCC/TGCC), indicating that the R486H does not measurably impact gamma-glutamyl carboxylase activity. The remaining two SNPs (2 and 5) are located within non-coding intron sequences. These 4 SNPs allowed for determining the genotype associated with the observed fatal coagulopathy. Screening for the premature truncation (SNP-4) based on the presence of a Bbv I restriction site in clinically normal lambs but not in the homozygous affected lambs allows for detection of the heterozygous state (GATT/GACC), because carrier animals are clinically normal.

A comparison of dobutamine infusion to exercise as a cardiac stress test in healthy horses.

This study was done to determine whether administration of dobutamine would produce echocardiographic and electrocardiographic alterations comparable to those induced by treadmill exercise in healthy horses. Fourteen horses received maximal treadmill exercise and, separately, intravenous dobutamine infusion up to a maximum rate of 50 microg/kg/min. Ten of the 14 horses were euthanized, and the myocardial tissues were examined grossly and histopathologically. No significant differences were found in the chronotropic effects of dobutamine and exercise (P = .905). Dobutamine induced greater interventricular septal thickening during systole (dobutamine = 4.78 cm, exercise = 4.03 cm; P = .004). and greater left ventricular diameters during diastole (dobutamine = 9.73 cm, exercise = 9.26 cm; P = .037), than did exercise treatment. Horses exhibited transient signs of sweating and restlessness during infusion of moderate to maximum doses of dobutamine. Ventricular ectopy seen in 11 of 14 horses was attributed to the arrhythmogenic properties of dobutamine, as well as to increased vagal tone present at low dobutamine doses. Myocardial lesions characteristic of catecholamine myotoxicity were present in 2 of the 10 horses examined. Although dobutamine induces chronotropic and inotropic changes similar to those induced by exercise, the use of high-dose dobutamine as a cardiac stressor in horses cannot be advocated because of potential development of arrhythmias or myotoxicity.

Atrial natriuretic peptide blockade exacerbates high altitude pulmonary edema in endotoxin-primed rats.

High altitude pulmonary edema (HAPE) is associated with increases in pulmonary arterial and hydrostatic pressures and an increase in pulmonary vascular permeability. There is evidence to suggest that inflammatory mediators may cause some forms of HAPE, and Salmonella enteritidis endotoxin (ETX) is known to activate neutrophils and inflammatory mediators, such as TNF-alpha and IL1-beta. Since HAPE has been produced in rats primed with ETX, we hypothesized that ANP release and action may ameliorate HAPE and that ANP blockade may exacerbate HAPE in ETX-primed rats exposed to high altitude (HA). Plasma ANP, right atrial ANP mRNA, and indexes of lung injury were measured in rats primed with endotoxin (ETX) (0.1 mg/kg BW, i.p.) and exposed to simulated HA (4267 m; P(B) = 440 mmHg) for either 12 or 24 h. Catheters were chronically inserted into the right carotid artery, pulmonary artery, and jugular vein for assessment of hemodynamic parameters in response to ETX and/or HA. In addition, some rats were injected with an antibody against ANP (alphaANP) prior to normoxic (NX) or HA exposure. Pulmonary arterial pressure increased in the alphaANP group (50 +/- 20%; p < or = 0.05) and in the HA + alphaANP (51 +/- 15%; p < or = 0.05) group at 12 h compared to NX sham rats injected with normal rabbit serum. In addition, systemic arterial pressure was significantly lower in the HA + ETX rats compared to HA + ETX + alphaANP rats (p < or = 0.001). Plasma ANP levels were significantly higher at 12 and 24 h in ETX, HA, and HA + ETX groups (p

Improved method for the synthesis of trans-feruloyl-beta-sitostanol.

Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels in humans by up to 15%, and at least two products, Benecol and Take Control, are now on the market as naturally derived fatty acid esters of phytostanols (stanol esters) and phytosterols (sterol esters), respectively. A synthetic process was developed to synthesize gram quantities of trans-feruloyl-beta-sitostanol from ferulic acid and beta-sitostanol, with high purity and yields of approximately 60%. The process involves (a) condensation of trans-4-O-acetylferulic acid with the appropriate phytostanol or phytostanol mixture in the presence of N,N-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, (b) separation of the trans-4-O-acetylferuloyl products by preparative liquid chromatography, (c) selective deacetylation of the feruloyl acetate, and (d) chromatographic purification of the feruloylated phytostanols. The process was successfully applied to synthesize stanol trans-feruloyl esters from "Vegetable Stanols", a mixture of approximately 70:30 beta-sitostanol and beta-campestanol, in comparable purity and yield.

Minimal incision rhytidectomy (short scar face lift) with lateral SMASectomy: evolution and application.

BACKGROUND: The evolution of the author's technique for minimal incision rhytidectomy is reviewed. OBJECTIVE: The purpose of this article is to outline the indications, advantages, and disadvantages of this technique. METHODS: A total of 749 cases covering more than 10 years of clinical experience are reviewed. A classification of patient types is proposed that includes indications and surgical programs appropriate for each patient category. RESULTS: In properly selected patients, the technique is safe, reliable, and reproducible. Complication rates are similar to those of other standard techniques. CONCLUSIONS: Minimal incision rhytidectomy with lateral SMASectomy is a useful technique that the plastic surgeon can add to his or her armamentarium.

Minimal incision rhytidectomy (short scar face lift) with lateral SMASectomy.

Minimal incision rhytidectomy has the primary advantages of preserving the posterior hairline and avoiding retroauricular scars. This short-scar face lift with lateral SMASectomy and platysma resection, performed by the author on more than 500 patients in the past 3 years, provides the versatility of traditional SMAS flap undermining and the safety and rapidity of SMAS plication.

Rate and equilibrium constants for the dehydration and deprotonation reactions of some monoacylated and glycosylated cyanidin derivatives.

The rate and equilibrium constants for the pH-dependent hydration of a series of monoacylated anthocyanins semi-biosynthesized using a wild carrot suspension culture have been measured. The features of the acyl groups that decrease the hydration and thus decrease the loss of color of these anthocyanins are the presence of the side-chain double bond of the cinnamic acids and the presence of electron-donating substituents at the para-position of the acyl group. Methoxyl groups in the meta-position of the acyl groups have a greater effect than can be explained by their effects on the electron density in the acyl group. The data also suggest that there may be additional features of the acyl groups which contribute to the color retention in anthocyanins.

Pulmonary thromboembolism in 29 dogs: 1985-1995.

Pulmonary thromboembolism (PTE) occurs as a complication to a number of commonly encountered clinical diseases. Antemortem recognition of this life-threatening disorder is hampered by nonspecificity of clinical signs. This retrospective study was performed to analyze clinical features, laboratory findings, imaging abnormalities, and concurrent postmortem diagnoses in 29 dogs with confirmed pulmonary embolism. A variety of clinicopathologic and radiographic abnormalities were noted but there were no pathognomonic findings for PTE. Arterial blood gas analyses were performed in 15 (52%) of 29 dogs; 12 (80%) of 15 exhibited hypoxemia and 15 (100%) of 15 had increased alveolar-arterial oxygen gradients. Response to supplemental O2 was variable and did not correlate with the presence or absence of additional pulmonary pathology on postmortem. At postmortem, 25 (86%) of 29 dogs had grossly visible emboli, 17 (59%) of 29 dogs had multiple disease processes, and 16 (55%) of 29 dogs had additional pulmonary pathology. PTE was suspected antemortem in 11 (38%) of 29 dogs. In dogs with respiratory signs consistent with PTE, the condition was a differential diagnosis in 11 of 17 animals; all had diseases previously reported to be associated with PTE. Neoplasia, systemic bacterial disease, and immune-mediated hemolytic anemia were diagnosed most frequently.

Hereditary deficiency of vitamin-K-dependent coagulation factors in Rambouillet sheep.

A flock of Rambouillet sheep experienced unexpected lamb mortality associated with excessive bleeding at the time of parturition. Most lambs died of blood loss through the umbilicus or into subcutaneous tissues. Subsequently, nine ewes which had previously delivered lambs that bled to death were bred to the suspected sire of the previous bleeding lambs. Fifteen lambs were born alive the following Spring, and three males and one female bled clinically. These lambs had markedly decreased factor IX (< 16%) and factor X (< 4%) activities, with variably decreased factor II (11-36%) and factor VII (20-37%) activities. Protein C chromogenic activity was also markedly decreased (< 1%) in these lambs. The results from crossed immunoelectrophoresis and 'protein-induced-in-vitamin-K-absence' determination of the plasma of affected lambs, with antiserum directed against coagulation factor X, protein C or proteins S, suggested that these proteins were not carboxylated normally. Examination of liver from one lamb in the first batch and the four subsequent lambs did not reveal a known vitamin K antagonist. The breeding data suggested that the coagulopathy in these sheep was inherited as an autosomal recessive trait. The genetic or molecular defect that exists in these lambs is unknown, but possibilities include abnormal gamma-glutamyl carboxylase activity or abnormal metabolism of vitamin K.

Synthesis and electrochemistry of anthraquinone-oligodeoxynucleotide conjugates.

Electroactive oligodeoxynucleotides (ODNs) with specific base sequences have a potential application as electrical sensors for DNA molecules. To this end, a phosphoramidite that bears a 9, 10-anthraquinone (AQ) group tethered to the 2'-O of the uridine via a hexylamino linker, 2'-O-[6-[2-oxo(9, 10-anthraquinon-2-yl)amino]hexyl]-5'-O-(4,4'-dimethoxytrityl)uridi ne 3'-[2-(cyanoethyl)bis(1-methylethyl)phosphoramidite] (3), has been synthesized and used to prepare three ODNs with tethered AQs using standard phosphoramidite chemistry. The synthetic methodology thus allows the synthesis of ODNs with electroactive tags attached to given locations in the base sequence. Cyclic voltammetric behavior of these AQ-ODN conjugates was examined in aqueous buffer solutions at a hanging mercury drop electrode. At slow sweep rates, nearly reversible two-electron waves characteristic of an adsorbed anthraquinone/hydroquinone redox couple was observed for all of the AQ-ODN conjugates. Approximate Langmuirian isotherms were found for the AQ-ODNs with molecular footprints, calculated from the saturation coverages, that scaled with molecular size. The cyclic voltammetric response of the duplexes formed from the AQ-ODNs and their complementary ODN was complicated by the competitive adsorption of the individual ODNs and possibly the duplex species as well.

A chiral rhodium complex for rapid asymmetric transfer hydrogenation of imines with high enantioselectivity.

[formula: see text] A chiral rhodium complex, (R)-Cp*RhCl[(1S,2S)-p-TsNCH(C6H5)CH(C6H5)NH2] (1a, (S,S)-Cp*RhClTsDPEN), generated from [Cp*RhCl2]2 and (1S,2S)-N-p-toluenesulfonyl-1,2-diphenylethylenediamine [(S,S)-TsDPEN], and its enantiomer 1b were found to provide superior catalysts for the rapid, high-yielding, asymmetric transfer hydrogenation of some heterocyclic imines, using an HCO2H-Et3N azeotrope as the hydrogen source.

Pedal osteosarcoma in a donkey.

An 18 1/2-year-old castrated male donkey with progressively worsening right forelimb lameness presented with a mass on the distal dorsal aspect of its P3 bone. Grossly, the firm, gritty mass was infiltrative, disrupted the contours of the overlying hoof wall, and had mottled and cavitated areas on cut surface. Histologically, the growth was composed of densely cellular sheets of mildly pleomorphic mesenchymal cells forming irregularly shaped islands of poorly mineralized osteoid. The neoplastic mass had patchy areas of necrosis. The diagnostic possibilities considered for this donkey's mass include osteosarcoma, osteoma, ossifying fibroma, and fibrous dysplasia. Careful consideration of the gross and histological characteristics of this donkey's mass support a diagnosis of osteosarcoma.

Evidence of a role for phosphatidylinositol 3-kinase activation in the blocking of apoptosis by polyomavirus middle T antigen.

A polyomavirus mutant (315YF) blocked in binding phosphatidylinositol 3-kinase (PI 3-kinase) has previously been shown to be partially deficient in transformation and to induce fewer tumors and with a significant delay compared to wild-type virus. The role of polyomavirus middle T antigen-activated PI 3-kinase in apoptosis was investigated as a possible cause of this behavior. When grown in medium containing 1D-3-deoxy-3-fluoro-myo-inositol to block formation of 3'-phosphorylated phosphatidylinositols, F111 rat fibroblasts transformed by wild-type polyomavirus (PyF), but not normal F111 cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI 3-kinase, stimulated apoptosis in PyF cells but not in normal cells. Activation of Akt, a serine/threonine kinase whose activity has been correlated with regulation of apoptosis, was roughly twofold higher in F111 cells transformed by either wild-type virus or mutant 250YS blocked in binding Shc compared to cells transformed by mutant 315YF. In the same cells, levels of apoptosis were inversely correlated with Akt activity. Apoptosis induced by serum withdrawal in Rat-1 cells expressing a temperature-sensitive p53 was shown to be at least partially p53 independent. Expression of either wild-type or 250YS middle T antigen inhibited apoptosis in serum-starved Rat-1 cells at both permissive and restrictive temperatures for p53. Mutant 315YF middle T antigen was partially defective for inhibition of apoptosis in these cells. The results indicate that unlike other DNA tumor viruses which block apoptosis by inactivation of p53, polyomavirus achieves protection from apoptotic death through a middle T antigen-PI 3-kinase-Akt pathway that is at least partially p53 independent.