Reversal of apixaban anticoagulation by four-factor prothrombin complex concentrates in healthy subjects: a randomized three-period crossover study.

Essentials Prothrombin complex concentrates (PCCs) may reverse the effect of factor Xa (FXa) inhibitors. We conducted an open-label, randomized, placebo-controlled, three-period crossover study in 15 subjects. Both PCCs rapidly reversed apixaban-mediated decreases in mean endogenous thrombin potential. Four-factor PCC administration had no effect on apixaban pharmacokinetics or anti-FXa activity. SUMMARY: Background Currently, there is no approved reversal agent for direct activated factor Xa (FXa) inhibitors; however, several agents are under investigation, including prothrombin complex concentrates (PCCs). Objective This open-label, randomized, placebo-controlled, three-period crossover study assessed the effect of two four-factor PCCs on apixaban pharmacodynamics and pharmacokinetics in 15 healthy subjects. Methods Subjects received apixaban 10 mg twice daily for 3 days. On day 4, 3 h after apixaban, subjects received a 30-min infusion of 50 IU kg-1 Cofact, Beriplex P/N (Beriplex), or saline. Change in endogenous thrombin potential (ETP), measured with a thrombin generation assay (TGA), was the primary endpoint. Secondary endpoints included changes in other TGA parameters, prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time, anti-FXa activity, apixaban pharmacokinetics, and safety. Results Apixaban-related changes in ETP and several other pharmacodynamic measures occurred following apixaban administration. Both PCCs reversed apixaban's effect on ETP; the differences in adjusted mean change from pre-PCC baseline to end of infusion were 425 nm min (95% confidence interval [CI] 219.8-630.7 nm min; P < 0.001) for Cofact, and 91 nm min (95% CI - 31.3 to 212.4 nm min; P > 0.05) for Beriplex. Both PCCs returned ETP to pre-apixaban baseline levels 4 h after PCC infusion, versus 45 h for placebo. For both PCCs, mean ETP peaked 21 h after PCC initiation, and then slowly decreased over the following 48 h. Both PCCs reversed apixaban's effect on TGA peak height, PT, and INR. Apixaban pharmacokinetic and anti-FXa profiles were consistent across treatments. Conclusions Cofact and Beriplex reversed apixaban's steady-state effects on several coagulation assessments.

Modeling effects of SGLT-2 inhibitor dapagliflozin treatment versus standard diabetes therapy on cardiovascular and microvascular outcomes.

AIMS: Dapagliflozin, a sodium-glucose cotransporter 2 (SGLT-2) inhibitor, has been shown to lower glycated hemoglobin (HbA1c), weight, blood pressure and serum uric acid in clinical trials. Plasma lipids were also evaluated as exploratory variables. The goal of this study was to estimate the long-term cardiovascular (CV) and microvascular outcomes of dapagliflozin added to the standard of care (SOC) versus SOC using simulation methodology. METHODS: The Archimedes Model, a validated model of human physiology, diseases and healthcare systems, was used to model a type 2 diabetes mellitus (T2DM) population derived from National Health and Nutrition Examination Survey (NHANES) with HbA1c 7-10%, taking a single oral antidiabetic agent [metformin, sulfonylureas SU or thiazolidinedione (TZD)] at the beginning of the trial. A 20-year trial was simulated comparing dapagliflozin 10 mg, given in addition to SOC, with SOC alone. SOC was based on American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD) 2012 guidelines and included diet, metformin, SU, TZD, dipeptidyl peptidase-4 (DPP-4), glucagon-like peptide-1 (GLP-1), and insulin therapies, with usage levels reflective of those in NHANES. Dapagliflozin effects were derived from phase 3 clinical trial results. End points included CV and microvascular outcomes. RESULTS: Over a 20-year period, patients on dapagliflozin were projected to experience relative reductions in the incidence of myocardial infarction (MI), stroke, CV death, and all-cause death of 13.8, 9.1, 9.6 and 5.0%, respectively, and relative reductions in the incidence of end-stage renal disease (ESRD), foot amputation, and diabetic retinopathy of 18.7, 13.0 and 9.8%, respectively, when compared with SOC. CONCLUSIONS: On the basis of simulation results, adding dapagliflozin to currently available treatment options is projected to further decrease the CV and microvascular complications associated with T2DM.

DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection.

We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (beta-galactosidase) transfection than SMC grown on native collagen (18.3+/-1.2 vs 1.0+/-0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-alpha(v)beta(3) integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by alpha(v)beta(3) integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-alpha(v)beta(3) antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0+/-1.8 vs 3.02+/-0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-alpha(v)beta(3) antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

Power spectral analysis of heart rate variability in rats as a quantitative tool in the PK-PD analysis of the parasympatholytic activity of atropine.

PURPOSE: To utilize power spectral analysis (PSA) of heart rate variability (HRV) as a pharmacodynamic (PD) measure of atropine parasympathetic effect, and to model the kinetics of action. METHODS: Heart rate data was collected following atropine administration to rats and was analyzed off-line for high frequency peaks by PSA of HRV as a measure of parasympathetic tone. A temporal cumulative approach (TCA) detected transient changes in parasympathetic activity. The pharmacokinetics (PK) was analyzed and linked to both direct and indirect PK-PD models. RESULTS: TCA enabled a quantitative measure of atropine parasympathetic activity. A simultaneous fit of the indirect PK-PD model to the experimental data of all three atropine doses successfully captured the experimental data. CONCLUSIONS: TCA can be used as a quantitative measure of parasympathetic tone. Our work has established a preclinical model to investigate the kinetics of drug action on the autonomic nervous system.

Cumulative plot of heart rate variability spectrum assesses kinetics of action of cholinergic drugs in rats.

A new approach to assess autonomic nervous system (ANS) activity and its response to drug action is presented. Our approach is based on the use of a cumulative plot of data obtained by power spectral analysis of heart rate variability, in defined frequency bands, during short time epochs (e.g., 2 min in rats). The substantial temporal variability in power evolving from the constant balancing nature of the ANS activity is minimized by this approach and produces a measurable index of ANS activity vs. time. The cumulative plot emphasizes the temporal response pattern of different components of the ANS and thereby facilitates the investigation of the kinetics of action of drugs affecting the ANS. We used this method to measure the activity of cholinergic drugs in freely moving Sabra rats. Bolus atropine doses between 0.5 and 2 mg/kg produced a similar magnitude of effect, reduction of the ascending slope by 0. 003 power units/h, whereas the duration of this effect was dose dependent. A lower atropine dose (0.1 mg/kg) or 0.5 mg/kg scopolamine elevated the slope (0.074 and 0.054 power units/h for 206 and 216 min, respectively). The method was used similarly to assess the interaction between cholinergic drugs. Pretreatment with pyridostigmine produced temporal blockage of the anticholinergic activity of atropine.

The effect of cimetidine on the pharmacodynamics of theophylline-induced seizures and ethanol hypnotic activity.

Due to its availability as an over-the-counter drug, the use of cimetidine is increasing, thus adverse interactions with other commonly used agents may also increase. The aim of this study was to investigate whether acute administration of cimetidine could alter the pharmacodynamics of theophylline neurotoxicity and the hypnotic action of ethanol. To examine these questions, rats received a dose of 77 mg/kg cimetidine followed by a constant infusion of either theophylline (1.2 mg/min.) or ethanol (16.3 mg/min.) until the onset of the pharmacological end point, maximal seizure or loss of righting reflex, where samples of blood and brain were obtained and assayed for either theophylline or ethanol. We report that cimetidine in doses that may cause pharmacokinetic interactions did not affect the concentration-effect relationship of either the stimulating action of theophylline or the depressant activity of ethanol. These outcomes emphasize the relative safety which patients using cimetidine in self-medication rely on.

The anticonvulsant effect of deprenyl on pentylenetetrazol-induced seizures in Lewis rats.

There is recent evidence that deprenyl may have anticonvulsant action in a rat kindling model of epilepsy as well as in a maximal electroshock model. We therefore investigated the effect of deprenyl on the brain sensitivity threshold to pentylenetetrazol (PTZ)-induced maximal seizures in Lewis rats, in a model that provides pharmacodynamic information free of pharmacokinetic interference. The novel finding of this investigation was the anticonvulsant effect of deprenyl following repetitive administration whereas a single deprenyl dose did not affect the PTZ concentrations required to induce maximal seizures. The data suggests that the mechanism of this effect is not associated with the dopaminergic activity of deprenyl since pretreatment with both bromocriptine (a dopamine D2 agonist) and haloperidol (dopamine antagonist) did not affect the seizure threshold, whereas levodopa caused a proconvulsant effect. It was also concluded that the mechanism is not related to changes in acetylcholine levels since prolonged pretreatment with deprenyl did not attenuate the brain sensitivity to pilocarpine-induced seizures. The fact that long term administration of deprenyl was needed to produce its anticonvulsant effect may indicate that the anticonvulsant effect of deprenyl may be due to changes in levels of certain endogenous compounds or down or up-regulation of relevant receptor/effector units.

The aging thyroid. I. A description of lesions and an analysis of their age and sex distribution.

Two types of follicular change in the thyroid are described. The first represents an involutional change in which colloid cysts (macrofollicles) are often found. The second represents a hyperplastic-dysplastic (microfollicular) lesion. Two types of change in calcitonin (C) cells are also described, one of which may represent an involutional process, although this is not certain. The second is a hyperplastic (micronodular) lesion, but without evidence of dysplasia. An infiltration of lymphocytes is also commonly encountered. Only about 15% of cases after age 60 are free of the follicular and lymphoid lesions. Both the macro- and microfollicular lesions show a statistically significant increase with age, as do the lymphoid lesion and its derivative Hashimoto's thyroiditis. C cell micronodules also appear to increase with age, but there are too few such cases for a reliable statistical analysis. An analysis of combinations of the follicular and lymphoid lesions show a particularly frequent association of the microfollicular and lymphoid lesions. Evidence is presented suggesting that many microfollicular lesions may represent clones of mutated cells, and that the lymphocytes may be targeted to these microfollicles. Lymphocytes were rarely found in association with C cell micronodules. Within individual thyroids there is considerable variation in the proportion of each lesion when combinations are present. Thus with advancing age there is an increasing heterogeneity of thyroid structure that may confound attempts to correlate structural and physiological changes with age.

The aging thyroid. II. An immunocytochemical analysis of the age-associated lesions.

This study utilizing immunocytochemical techniques consists of two segments, 1) an analysis of the changes with age in the hormone content of normal, involuting, macro- and microfollicles, as well as calcitonin (C) cells, and 2) an analysis of the components of the lesion characteristic of lymphocytic thyroiditis. Involuting and macro- (cystic) follicles show a decline in hormone content with age, while microfollicles show a variable pattern with some showing a high and others a low content, as well as some showing an absence of hormone. There also appears to be an increase with age in hormone-containing C cell micronodules, but we were unable to demonstrate a decline in hormone content of individual C cells. Studies on the in vivo binding of immunoglobulins, as an indicator of thyroid antibody binding, showed an increase with age in IgG and IgM binding to follicular epithelium. Much stronger reactions were observed in the epithelium of microfollicles than in that of involuting or macrofollicles. There were also examples of presumptive in vivo antibody binding by C cell micronodules. We were unable to demonstrate an imbalance of helper/suppressor T cell ratio in the thyroiditis lesion, but did find that some lymphocytes contain thyroid hormones, suggesting that the latter may act as cytokines and assist in the perpetuation of the lesion. The epithelial expression of human lymphocyte antigens (HLA) was strong in microfollicles and weak or absent in involuting or macrofollicles in sections with lymphocytic thyroiditis. It was also strong in the epithelium of a microfollicular adenoma without a lymphocytic infiltrate while weak or absent in the epithelium of adjacent normal follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone levels during glucose tolerance test in euthyroid subjects.

Twelve normal, healthy, clinically and biochemically proven euthyroid volunteer subjects (age 19-35) were administered a standard glucose tolerance test (100 g glucose orally) and thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) levels were determined by specific radioimmunoassays to determine the acute effect of glucose and insulin on peripheral monodeiodination of thyroxine. The fasting levels of T4, T3, rT3 and free thyroxine were 82.4 nmol/l, 1.7 nmol/l, 0.52 nmol/l, and 0.22 nmol/l, respectively, and these levels were unchanged during the 3 hours post-glucose load. The rise and fall of glucose and insulin levels were typical of the standard responses normally observed in the glucose tolerance test. The variations in insulin and glucose levels were not correlated with thyroid hormone concentrations at any interval during the test. It is therefore concluded that dietary glucose does not acutely cause shifts in peripheral monodeiodination of thyroxine in healthy euthyroid subjects.

Effect of pregnancy on thyroxine binding globulin (TBG) in partial TBG deficiency.

An unusual opportunity was afforded to study the effect of endogenous increase in estrogen on thyroxine binding globulin (TBG) throughout pregnancy in a partially TBG-deficient female who conceived subsequent to initial examination. TBG binding of 125I-thyroxine (T4) before and up to four months of pregnancy was low in comparison to normal. Starting from six months and up to the end of pregnancy, TBG activity showed a definite increase although still below normal. TBG binding capacity increased from a low value of 4.2 microgram T4/100 ml ,efore pregnancy to a value 9.3 microgram T4/100 ml in the last month of pregnancy. This was accompanied by an increase in immunoassayable TBG from less than 1 mg/100 ml in the second month of pregnancy to 1.9 mg/100 ml in the last month, and an increase of T4 from 3.1 microgrom/100 ml to 4.3 microgram/100 ml. Two weeks after delivery, TBG binding of 125I-T4 showed a precipitous decline to the abnormally low distribution noted prior to and during the early months of pregnancy. TBG binding activity was normal in the cord blood of the infant. These studies provide the first direct evidence that increase in endogenous estrogen results in detectable increases in TBG concentration in the human with partial TBG deficiency.

Studies on obesity. III. effect of triiodothyronine (T3) on thyroglobulin autoantibodies in euthyroid obese subjects.

Effect of T3 therapy on tanned red cell agglutinating thyroglobulin (TRC-TG) antibodies in 10 obese subjects without apparent thyroid disease was investigated. Six other obese subjects without thyroid dysfunction and of approximately the same mean age who also had circulating TRC-TG antibodies served as control subjects and were untreated. In vitro thyroid tests (TSH, total and free T4) performed before T3 therapy, as well as clinical examination, showed thyroid function to be normal in all subjects, and there was no evidence of thyroiditis. TRC-TG antibodies were present in low to moderate titers of 40-1280 in control subjects as well as in subjects selected for T3 treatment. Therapy with T3 was started at 50 mug/day and gradually increased to a maximum of 250 mug/day, depending on clinical needs. T3-treated as well as untreated obese control subjects were all maintained on a high protein, low fat, low carbohydrate diet. Duration of T3 therapy varied from 2-8 mo, and in all but one T3-treated subject, TRC-TG antibodies completely disappeared. In the one exceptional case, TRC-TG antibody titer decreased from 1280 to 80 after 7 mo of therapy. In non-T3-treated obese control subjects, antibody titers remained at the same levels throughout the observation period, thereby indicating a lack of spontaneous regression of circulating immune response. Therapy with T3, by inhibiting TSH, may have caused regression of inapparent immunologic thyroid lesion, thus leading to the disappearance of circulating TRC antibodies; alternatively, T3 specifically may have accelerated catabolism of thyroid antibodies. The latter possibility is favored in the absence of clinical and laboratory evidence of thyroiditis in T3-treated subjects.

Increased free thyroxine in a euthyroid patient with thyroxine-binding globulin deficiency.

An unusual finding of elevated free thyroxine (FT4) concentration coexistent with familial low thyroxine-binding globulin (TBG) is described in a euthyroid patient. Clinical and other laboratory tests were normal and consistent with a clinical diagnosis of euthyroidism. Low TBG (TBG capacity 3-10 mug/100 ml) was confirmed by various electrophoretic procedures and by TBG immunoassay (0.9 mg/100 ml). Serum T4 concentration was low (2.0-5.5 mug/100 ml). Triiodothyronine (T3) resin uptake was increased (88-93%) and the free thyroxine index was normal. Twenty-four-hour thyroidal 131I uptake was normal and varied between 13-20%. Serum thyroid-stimulating hormone (less than 2 muunits) and T3 concentration (80 ng/100 ml) were also normal. Long-acting thyroid stimulator (LATS) was not present. Free T4 concentration measured by dialysis procedure was markedly elevated (9.2-12.1 ng/100 ml). Pulse T3 and T4 tracer kinetic studies by the single compartmental method revealed accelerated blood disappearance of T4 (t 1/2 = 3.7 days) but normal disappearance of T3 (t 1/2 = 2.1 days). Extrathyroidal hormonal iodine pool (569 mug) was in the range of normals. T4 degradation rate (144 mug/70 kg/day) was moderately elevated over the euthyroid mean values, but was still close to or within the upper normal range. T3 degradation was normal (34 mug/day). Failure to develop hyperthyroidism in the presence of elevated free T4 levels, but with normal T3 concentration and dynamics, may suggest that T3, not T4, plays a major role in regulating metabolic activity; alternatively, euthyroidism in this low-TBG patient despite elevated free T4 concentration may simply be explained on the basis of a daily T4 disposal rate which was not clearly abnormal.