Pharmacokinetics and anticoagulant properties of the factor VIIa-tissue factor inhibitor recombinant Nematode Anticoagulant Protein c2 following subcutaneous administration in man. Dependence on the stoichiometric binding to circulating factor X

Recombinant Nematode Anticoagulant Protein c2 (rNAPc2) is a potent (K(i) =10 pM), inhibitor of the factor VIIa/tissue factor (fVIIa/TF) complex that requires the prerequisite binding to zymogen or activated factor X (fX). In two double blind, place-bo-controlled, sequential dose-escalation phase I studies, rNAPc2 was found to be safe and well tolerated following single and repeat subcutaneous administrations in healthy human male volunteers at doses ranging from 0.3 to 5 micro g/kg. There was a dose-dependent elevation of the prothrombin time reaching almost 4-fold above the baseline value in the highest dose group that directly correlated with rNAPc2 plasma concentration. In contrast, there was little or no effect on the activated partial thromboplastin time, thrombin time or template bleeding time. The pharmacokinetic behavior of rNAPc2 revealed a dose-independent and prolonged elimination half-life (t(1/2)beta) with a mean of >50 hours. A high affinity interaction between rNAPc2 and plasma fX was shown to be essential for the prolonged t(1/2)beta in man using crossed immunoelectrophoresis and was confirmed by exploiting the considerably weaker interaction between rNAPc2 and bovine fX which resulted in an attenuated t(1/2)beta of approximately 1.5 hours in calves. The accumulated data suggests that rNAPc2 is safe and well tolerated following repeat subcutaneous administrations at doses up to 5 micro g/kg in healthy volunteers. In addition, the in vivo fate of rNAPc2 in man appears to be governed by its high affinity interaction with circulating fX. This data supports the continued development of this novel anticoagulant for the prevention and treatment of acute thrombotic disorders.     

Anticoagulant and anti-platelet effects are maintained following coadministration of otamixaban, a direct factor Xa inhibitor, and acetylsalicylic acid

The pharmacokinetics, pharmacodynamics and safety of the direct factor Xa inhibitor, otamixaban, with and without concomitant acetylsalicylic acid (ASA) were investigated in healthy volunteers. The study was a double-blind, placebo-controlled 3-way crossover study. Sixty-eight male volunteers in total were randomised to otamixaban, ASA, or otamixaban with ASA. ASA (300 mg once a day) was started 2 days before and continued on the day of the otamixaban 6-hour IV infusion (0.3 and 0.5 mg/kg). Pharmacokinetic and pharmacodynamic parameters (coagulation markers, platelet function tests and skin bleeding time) were determined. Drug interaction was assessed by the ratios of geometric means and 90% confidence intervals (90% CI)of the parameter estimates. Pharmacokinetic parameters of otamixaban remained unchanged with ASA. Ratios of geometric means (90% CI) were for Ceoi 96.54 (91.21-102.19) and 95.04 (90.10-100.24) and for AUC 98.0 (93.92-102.25) and 95.90 (92.61-99.31), for 0.3 and 0.5 mg/kg, respectively. No drug interaction was observed between otamixaban and ASA on the coagulation and platelet function parameters. Neither otamixaban nor ASA had an effect on skin bleeding time; their co-administration led to a slight prolongation of skin bleeding time above the normal range without any clinically relevant bleeding. This study demonstrated that the desired effects of otamixaban and ASA, namely anticoagulation and platelet inhibition, respectively, are maintained during co-administration of both drugs.     

Disposition of progabide and valproic acid following intraperitoneal administration in rhesus monkey

The pharmacokinetic characteristics of progabide, a new gamma-aminobutyric acid-mimetic drug, were evaluated following single- and multiple-dose administration of a progabide suspension through a chronic intraperitoneal catheter. Several issues pertaining to the bioavailability of progabide were addressed: first-pass effect, incomplete dissolution, and dose and time dependency. Four male monkeys received five treatments: three intraperitoneal doses (50, 100, and 150 mg), one oral dose (50 mg), and one intravenous bolus dose (150 mg). Bioavailability following intraperitoneal administration was incomplete, consistent with a first-pass effect predicted from intravenous data. There were no significant differences between the absolute bioavailabilities of the three intraperitoneal doses, which ranged between 40 and 49%. The apparent half-life (t 1/2) observed after intraperitoneal administration was significantly longer than the elimination half-life by the intravenous route and tended to increase with dose. This behavior is consistent with dissolution rate-limited absorption. The bioavailability of the suspension administered orally was compared with that of the intraperitoneal route, and no difference was found. However, the apparent t 1/2 by the oral route was significantly longer than that of the intraperitoneal route. In multiple-dosing studies, four different dosing regimens (all intraperitoneal) were examined, including 50 mg every 2 or every 6 h, 20 mg every 2 h, and 40 mg every 4 h. In all these regimens, plasma levels exhibited an accumulation compared with single-dose predictions. Large oscillations in plasma levels were observed when the dosing interval was 6 h and side effects were observed when the dosing interval was 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticoagulant pharmacodynamics of tinzaparin following 175 iu/kg subcutaneous administration to healthy volunteers

Tinzaparin, a sodium salt of a low-molecular-weight heparin (LMWH) produced via heparinase digestion, is used for the treatment of deep vein thrombosis (DVT) and pulmonary embolism in conjunction with warfarin for the prevention of DVT in patients undergoing hip or knee replacement surgery, and as an anticoagulant in hemodialysis circuits. Its average molecular weight ranges between 5500 and 7500 daltons (Da); the percentage of chains with molecular weight lower than 2000 Da is not more than 10% in the marketed tinzaparin formulation. While this fraction is generally considered pharmacologically inactive, this has never been evaluated in vivo. The importance of the < 2000 Da fraction on the anticoagulant pharmacodynamics of tinzaparin assessed by anti-Xa and anti-IIa activity was studied in a two-way crossover trial. In this trial, 30 healthy volunteers received a single 175 IU/kg subcutaneous administration of tinzaparin containing approximately 3.5% of the < 2000 Da fraction and a tinzaparin-like LMWH containing 18.3% of the < 2000 Da fraction. The anti-Xa/anti-IIa ratios of the drug substances were comparable at 1.5 and 1.7 for tinzaparin and the tinzaparin-like LMWH, respectively. Both formulations were safe and well tolerated. Mean maximum plasma anti-Xa activity (A(max)) was approximately 0.818 IU/ml at 4 h following tinzaparin injection. Mean maximum plasma anti-IIa activity was 0.308 IU/ml at 5 h postdose. Intersubject variation was lower (< 18% for both anti-Xa and anti-IIa metrics) than in previous fixed-dose administration studies. There was no correlation between anti-Xa or anti-IIa AUC or A(max) and bodyweight in the present study supporting the weight-adjusted dosing regimen. Individual anti-Xa and anti-IIa profiles following the single 175 IU/kg subcutaneous administration of the tinzaparin-like LMWH were similar to that obtained with tinzaparin. Based on average equivalence criteria, the two LMWH preparations were determined to be bioequivalent using either anti-Xa or anti-IIa activity as biomarkers. The calculated intrasubject variabilities were low (< 14% for anti-Xa activity and < 18% for anti-IIa activity) yielding little evidence for a significant Subject x Formulation interaction. In summary, anti-Xa and anti-IIa activity following a single subcutaneous administration of tinzaparin 175 IU/kg to healthy volunteers yielded activity consistent with targeted therapeutic levels derived from previous trials in adult DVT patients. Weight-based dosing for the treatment of DVT appears rational based on the reduction in anti-Xa and anti-IIa variability consistent with the recommendation derived from earlier fixed-dose pharmacokinetic studies. Furthermore, differences in the percentage of molecules in the < 2000 Da molecular weight fraction of tinzaparin do not translate into differences in anti-Xa and anti-IIa activity in vivo.     

Comparison of antithrombotic efficacy between edoxaban, a direct factor Xa inhibitor, and fondaparinux, an indirect factor Xa inhibitor under low and high shear rates

Edoxaban is an oral, direct factor Xa (FXa) inhibitor under late-phase clinical development. This study compared the antithrombotic efficacy of edoxaban with that of an indirect FXa inhibitor, fondaparinux, in in vivo venous and arterial thrombosis models and in ex vivo perfusion chamber thrombosis model under low and high shear rates in rats. Venous and arterial thrombi were induced by platinum wire insertion into the inferior vena cava and by application of FeCl? to the carotid artery, respectively. The perfusion chamber thrombus was formed by blood perfusion into a collagen-coated capillary at 150 s?1 (low shear rate) and 1,600 s?1 (high shear rate). Effective doses of edoxaban that reduced thrombus formation by 50% (ED??) in venous and arterial thrombosis models were 0.076 and 0.093 mg/kg/h, respectively. In contrast, ED?? of fondaparinux in the arterial thrombosis model (>10 mg/kg/h) was markedly higher compared to ED?? in the venous thrombosis model (0.021 mg/kg/h). In the perfusion chamber thrombosis model, the ratio of ED?? under high shear rate (1.13 mg/kg/h) to that under low shear rate (0.63 mg/kg/h) for edoxaban was 1.9, whereas that for fondaparinux was more than 66. While the efficacy of fondaparinux markedly decreased in arterial thrombosis and in a high-shear state, edoxaban exerted consistent antithrombotic effects regardless of flow conditions. These results suggest that shear rate is a key factor in different antithrombotic effects between edoxaban and fondaparinux.     

Regeneration of the magnocellular system of the rhesus monkey following hypothalamic lesions.

The hypothalamic magnocellular system of the rhesus monkey was studied with specific immunocytochemical techniques in animals that had undergone hypothalamic lesions. The results indicate that this system maintains a regenerative capacity even when its tracts are interrupted within the hypothalamus. New neurohemal units are reconstituted from newly formed vessels within the scar as well as from preexistent blood vessels, such as perforating and pial arterioles, and the vessels of the pars tuberalis of the pituitary gland, which normally do not contain neurosecretory terminals.     

Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis

An in vivo thromboplastin (TP)-induced venous stasis thrombosis model in rabbits was used to compare the efficacy of standard heparin with the selective factor Xa inhibitors, recombinant tick anticoagulant peptide (rTAP) and recombinant antistasin (rATS), in prophylactic prevention of thrombus formation. Heparin significantly reduced TP-induced clot formation at doses of 55 and 100 U kg-1h-1 yielding clot weights of 9 +/- 4 and 6 +/- 2%, respectively. Clot formation was significantly decreased by i.v. infusions of rTAP at doses of 21, 37 and 64 micrograms kg-1 min-1 resulting in normalized clot weights of 13 +/- 3, 8 +/- 2 and 2 +/- 1%, respectively. rATS was approximately 10-fold more potent than rTAP, reducing normalized clot weights to 16 +/- 5, 2 +/- 1 and 1 +/- 0.8% at rATS doses of 1.25, 2.5 and 5.0 micrograms kg-1 min-1, respectively. These data suggest that factor Xa-mediated inhibition of coagulation with rTAP and rATS is as effective as conventional anticoagulant treatment with heparin in preventing venous thrombosis.     

Face recognition in the rhesus monkey.

Rhesus monkeys were trained on a variety of simultaneous two-choice visual discrimination tasks to assess their ability to utilize pictures of other rhesus monkey faces as discriminative stimuli. The results revealed that this non-human primate is particularly adept at making such discriminations and is not confused by manipulations in orientation; posture; size; color; or illumination.     

Compound stimulus differentiation behavior in the rhesus monkey following periarcuate ablations

Anatomical and physiological investigations in cat and monkeys have demonstrated the existence of association areas where evoked and unit responses associated with more than one sensory modality can be recorded. Although it is often assumed that these areas of association cortex from the anatomical substratum for complex cognitive and integrative behavior in higher mammals, there is little experimental evidence in non-human primates to support this viewpoint. This experiment investigated the effects of periarcuate ablations on the ability of the rhesus monkey to perform a compound stimulus (lightand tone) versus component stimuli (lightor tone) differentiation. Ablation of this well-known polysensory area in the frontal lobe led to large peristent impairments in performance on this task, while the ability to peform intramodal and intermodal differentiations was spared. Ablations of comparable size in other parts of the frontal lobe had no affect on the performance of these tasks. It was concluded that one function of the periarcuate region may be to synthesize multiple sources of sensory input occuring simultaneously into meaningful elements.     

Increased brain GABA concentrations following acute administration of a selective serotonin reuptake inhibitor

OBJECTIVE: The authors used magnetic resonance spectroscopy (MRS) to assess the effect of acute administration of the selective serotonin reuptake inhibitor (SSRI) citalopram on cortical levels of gamma-aminobutyric acid (GABA). METHOD: Ten healthy volunteers received either intravenous citalopram (10 mg) or saline in a randomized, double-blind, crossover design. The occipital GABA/creatine ratio was measured with a proton MR spectral editing technique. RESULTS: In comparison with saline, citalopram produced a mean increase of 35% in relative brain GABA concentration in the occipital cortex. CONCLUSIONS: These findings extend previous work showing that SSRI treatment increases cortical GABA in depressed patients and suggest that this results from an action of SSRIs on GABA neurons rather than as a secondary consequence of mood improvement.     

Recombinant tissue factor pathway inhibitor enhances the binding of factor Xa to human monocytes

Tissue factor pathway inhibitor (TFPI) is a kunitz-type inhibitor of activated factor X (Xa). TFPI was reported to mediate Xa binding to a few of carcinoma cell lines. In this study it was observed that the Xa activity associated with human peripheral blood mononuclear cells (PBMC) incubated with Xa in the presence of recombinant TFPI (rTFPI) was much higher than with Xa alone. Xa activity on PBMC was also observed after whole blood was incubated with pre-formed Xa/TFPI complex. Further studies with flow cytometric analysis demonstrate that rTFPI enhances the binding of Xa to human monocytes. Western blot analysis showed that rTFPI was cleaved into a few of fragments after its incubation with monocytes either in the presence or absence of Xa. Based on these results and the observations reported by others, we speculate that Xa/TFPI complex may bind to human monocytes by a yet unidentified mechanism. The recovery of Xa activity from Xa/TFPI complex on PBMC may be related to the cleavage of rTFPI by Xa and/or monocyte proteases. This observation suggests a new mechanism by which monocytes become procoagulant in some pathological conditions in addition of the well known tissue factor expression on proinflammatic monocytes.     

Comparison of PD0348292, a selective factor Xa inhibitor, to antiplatelet agents for the inhibition of arterial thrombosis

The objective of this study was to determine if orally-administered PD0348292, a direct specific factor Xa inhibitor, inhibits thrombosis following porcine carotid arterial injury comparably to aspirin or clopidogrel alone or in combination. We further sought to determine whether the antithrombotic efficacy in vivo could be predicted using an ex-vivo perfusion chamber. Oral treatments included: PD0348292 (0.4, 0.9, or 4.3 mg/kg); PD0348292 (0.4 mg/kg) plus aspirin (325 mg); aspirin; clopidogrel (75 mg); aspirin plus clopidogrel; or vehicle (n = 6-10/group). Aspirin and clopidogrel were administered 27 and four hours pre-injury and PD0348292 or vehicle was administered four hours pre-injury. Both carotid arteries were crush-injured, and thrombus was measured by detection of (111)In-platelets over 30 minutes. Prior to injury, the antithrombotic efficacy was assessed by ex-vivo perfusion chamber platelet deposition. PD0348292 produced dose-dependent prothrombin time (0.9- to 2.9-fold) and aPTT (1.4- to 2.5-fold) prolongations. Bleeding times were significantly prolonged in each active drug group compared to vehicle, but were not significantly different between drug groups. PD0348292 significantly inhibited arterial platelet deposition (x10(6)/cm(2)) at 4.3(549 +/- 1,066), 0.9 (399 +/- 162) and 0.4 mg/kg (531 +/- 470) compared to vehicle (2,242 +/- 1,443). Aspirin (992 +/- 973), clopidogrel (537 +/- 483), clopidogrel plus aspirin (228 +/- 66) or PD0348292 plus aspirin (558 +/- 317) also significantly inhibited platelet deposition, although these values were not significantly different than with any dose of PD348292. Perfusion chamber platelet deposition correlated significantly with in-vivo anti-thrombotic response. In conclusion, PD0348292 inhibited arterial thrombosis comparable to aspirin plus clopidogrel. Perfusion chamber methodology may be useful in predicting in-vivo antithrombotic efficacy.     

Long-term effects of repeated methylamphetamine administration on monoamine neurons in the rhesus monkey brain

Previous studies indicate that the repeated administration of D-methylamphetamine (MA) produces a long-lasting depletion of dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) in various brain regions of a number of species. The objectives of the present study were: (1) to establish a short, subcutaneous injection regimen which would reliably produce the neuronal alterations; (2) to evaluate MA-induced NE depletions produced by this new regimen; and (3) to determine whether central MA-induced neuronal changes are reflected in changes in cerebrospinal fluid monoamine metabolite concentrations. It was observed that high doses of MA administered (s.c.) over a 2-week period to rhesus monkeys produced decreases in DA and 5-HT, but not NE levels, in various brain regions. The decrease in caudate DA levels was accompanied by a decrease in the number of DA uptake sites, a decrease in the level of homovanillic acid (HVA) and an increase in DA turnover. This decrease in brain DA was also accompanied by a decrease in the cerebrospinal fluid concentration of HVA.     

Axonal sprouting of a brainstem-spinal pathway after estrogen administration in the adult female rhesus monkey

The nucleus retroambiguus (NRA) is located in the caudal medulla oblongata and contains premotor neurons that project to motoneuronal cell groups in the brainstem and spinal cord. NRA projections to the lumbosacral cord are species specific and might be involved in mating behavior. In the female cat, this behavior is estrogen dependent, and estrogen induces axonal sprouting in the NRA-lumbosacral pathway. Because female receptive behavior in primates is not fully dependent on estrogen, the question arises as to whether the capacity of estrogen-induced sprouting is preserved in primates. The effect of estrogen was studied on the NRA-lumbosacral projection with the use of wheat germ agglutinin conjugated to horseradish peroxidase as a tracer in six adult ovariectomized rhesus monkeys with or without estrogen priming (three controls and three treated with 20 microg/day of estradiol benzoate subcutaneously for 14 days). Light microscopy showed that the density of arborizing labeled NRA axons in the lumbosacral cord was greater in estrogen-treated than in control animals. Ultrastructurally, labeled NRA terminal profiles were quantified in motoneuron pools that supply muscles of the abdominal wall, axial, and pelvic floor. After estrogen treatment, the average number of labeled terminal profiles per area of the abdominal wall, axial, and pelvic floor motoneuron pool increased 1.5-, 3.3-, and 2.8-fold, respectively. In the estrogen-treated cases, 8.9% of labeled terminal profiles showed characteristics of growth cones. In controls, such profiles were rarely observed. The results showed that estrogen induces axonal sprouting in a brainstem-spinal pathway in the adult female rhesus monkey. These findings supported the concept that the NRA-lumbosacral pathway may be involved in sexual behavior. Moreover, they demonstrated that a long descending brainstem-spinal tract in adult nonhuman primates retains the capacity for axonal sprouting.     

Neurogenesis of the amygdaloid nuclear complex in the rhesus monkey.

The time course of neurogenesis for neurons which comprise the amygdaloid complex in Rhesus monkeys was determined using tritiated thymidine autoradiography. Fourteen pregnant monkeys received injections of tritiated thymidine between embryonic days 27 (E27) and 56 of their 165 day gestation and offspring were sacrificed during the early postnatal period. The first neurons destined for the amygdaloid complex were generated at E33 making them among the earliest postmitotic neurons in the telencephalon. Neurogenesis peaked within all nuclei of the amygdaloid complex between E38 and E48 and had ceased between E50 and E56. While amygdaloid neurogenesis in postnatally sacrificed monkeys displayed a dorsal-to-ventral gradient of radiolabeled neurons, the considerable rotation of the temporal lobe during the latter stages of primate development indicates that neurogenesis in the embryo, during the first third of gestation, actually occurs across a medial-to-lateral gradient. This medial-to-lateral gradient occurs as a smooth wave across the amygdaloid nuclei and does not respect neuroanatomical subdivisions or patterns of connectivity of the amygdaloid nuclei in the Rhesus monkey.