Preclinical and clinical pharmacology of TPA023B, a GABAA receptor α2/α3 subtype-selective partial agonist.

In the accompanying paper we describe how MRK-409 unexpectedly produced sedation in man at relatively low levels of GABA(A) receptor occupancy (∼10%). Since it was not clear whether this sedation was mediated via the α2/α3 or α1 GABA(A) subtype(s), we characterized the properties of TPA023B, a high-affinity imidazotriazine which, like MRK-409, has partial agonist efficacy at the α2 and α3 subtype but is an antagonist at the α1 subtype, at which MRK-409 has weak partial agonism. TPA023B gave dose- and time-dependent occupancy of rat brain GABA(A) receptors as measured using an in vivo [(3)H]flumazenil binding assay, with 50% occupancy corresponding to a respective dose and plasma drug concentration of 0.09 mg/kg and 19 ng/mL, the latter of which was similar to that observed in mice (25 ng/mL) and comparable to values obtained in baboon and man using [(11)C]flumazenil PET (10 and 5.8 ng/mL, respectively). TPA023B was anxiolytic in rodent and primate (squirrel monkey) models of anxiety (elevated plus maze, fear-potentiated startle, conditioned suppression of drinking, conditioned emotional response) yet had no significant effects in rodent or primate assays of ataxia and/or myorelaxation (rotarod, chain-pulling, lever pressing), up to doses (10 mg/kg) corresponding to occupancy of greater than 99%. In man, TPA023B was well tolerated at a dose (1.5 mg) that produced occupancy of >50%, suggesting that the sedation previously seen with MRK-409 is due to the partial agonist efficacy of that compound at the α1 subtype, and highlighting the importance of antagonist efficacy at this particular GABA(A) receptor population for avoiding sedation in man.

In vivo labeling of endothelin receptors with [(11)C]L-753,037: studies in mice and a dog.

UNLABELLED: Endothelin (ET) is a potent mammalian vasoconstrictive peptide and a pressor agent. Its 3 isoforms, ET-1, ET-2, and ET-3, mediate several physiologic actions in several organ systems, binding to 2 major receptor subtypes: ET(A) and ET(B). This study was undertaken to evaluate [(11)C]L-753,037 [(+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxy-propyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno [1,2-beta]pyridine-6-carboxylate), a new mixed ET receptor A and B antagonist, as a tracer for in vivo labeling of ET receptors in mice and a dog. METHODS: [(11)C]L-753,037 was synthesized, purified, and formulated from a normethyl precursor, L-843,974, and [(11)C]H(3)I. The tracer was studied for its in vivo kinetics, biodistribution, and ET receptor binding characteristics in mice. In the dog, PET imaging was performed to evaluate binding of [(11)C]L-753,037 to ET receptors in the heart. Specificity of binding was studied in the heart with the selective ET(A) antagonist L-753,164. RESULTS: Kinetic studies in mice showed highest tracer uptake at 5 min after injection in liver (25.0 percentage injected dose per gram [%ID/g]), kidneys (18.7 %ID/g), lungs (15.2 %ID/g), and heart (5.6 %ID/g). Initial high uptake in liver, lungs, and kidneys was followed by rapid washout during the next 10 min and a very slow clearance during the time of observation (2 h after injection). By contrast, the radioactivity in the heart remained constant over 2 h. Administration of both ET(A) (L-753,164) and mixed ET(A)/ET(B) (L-753,137) receptor antagonists resulted in dose-dependent inhibition of [(11)C]L-753,037 binding in mouse heart, lungs, and kidneys but not in the liver. Radioactivity in the brain was very low, indicating that the tracer does not cross the blood-brain barrier. In the dog, a dynamic PET study of the heart showed high tracer accumulation at 55-95 min after injection. Injection of L-753,164 at 30 min before [(11)C]L-753,037 administration led to a significant reduction in tracer binding. [(11)C]methyl triphenyl phosphonium was used as a tracer for reference images of the dog heart muscle. CONCLUSION: The results suggest that [(11)C]L-753,037 binds to ET receptors in vivo and is, therefore, a promising candidate for investigation of these receptors and their occupancy by ET receptor antagonists using PET.

Non-invasive radiotracer imaging as a tool for drug development.

Non-Invasive Radiotracer Imaging (NIRI) uses either short-lived positron-emitting isotopes, such as 11C and 18F, for Positron Emis ion Tomography (PET) or single photon emitting nuclides, e.g., 123I, which provide images using planar imaging or Single-Photon Emission Computed Tomography (SPECT). These high-resolution imaging modalities provide anatomical distribution and localization of radiolabeled drugs, which can be used to generate real time receptor occupancy and off-rate studies in humans. This can be accomplished by either isotopically labeling a potential new drug (usually with 11C), or indirectly by studying how the unlabelled drug inhibits specific radioligand binding in vivo. Competitive blockade studies can be accomplished using a radiolabeled analogue which binds to the site of interest, rather than a radiolabeled version of the potential drug. Imaging, particularly PET imaging, can be used to demonstrate the effect of a drug through a biochemical marker of processes such as glucose metabolism or blood flow. NIRI as a development tool in the pharmaceutical industry is gaining increased acceptance as its unique ability to provide such critical information in human subjects is recognized. This section will review recent examples that illustrate the utility of NIRI, principally PET, in drug development, and the potential of imaging advances in the development of cancer drugs and gene therapy. Finally, we provide a brief overview of the design of new radiotracers for novel targets.

Positron emission tomography neuroreceptor imaging as a tool in drug discovery, research and development.

Improved communication and cooperation between research-driven drug companies and academic positron emission tomography (PET) centers, coupled with improvements in PET camera resolution, the availability of small animal PET cameras and a growing list of neuroreceptor-specific PET tracers, have all contributed to a substantial increase in the use and value of PET as a tool in central nervous system drug discovery and development.

Radioiodinated endothelin-1: a radiotracer for imaging endothelin receptor distribution and occupancy.

Endothelin (ET) is one of the most potent vasoconstrictors known. Recently, ET has been implicated in various diseases, e.g., acute renal failure and congestive heart failure, which present the possibility of treating such diseases with endothelin receptor antagonists. However, establishing the dosages for these antagonists may be difficult because no convenient physiologic indicator of action exists, and because of complexities in receptor function. Two receptor subtypes have been identified for which selective antagonists have been reported (e.g., BQ-123 for the ETA receptor and BQ-788 for the ETB receptor). Of the three natural peptide hormones (ET-1, ET-2, and ET-3), ET-1 exhibits high affinity for both subtypes of receptor. Using the selective peptide antagonists, and a nonpeptide antagonist with relatively balanced affinity for the two subtypes (L-749,329), we have characterized the interactions of [125I]ET-1 with its receptors in vivo (in rat). BQ-123, BQ-788, and L-749,329 inhibited binding consistent with binding to a single receptor site. However, the sum of inhibition by the selective antagonist was greater than 100% (as defined by inhibition with L-749,329), which suggests (a) lower in vivo selectivity than determined in vitro and/or (b) receptor subtype interactions. The latter explanation is supported, in part, by in vitro autoradiographic studies as well as studies in isolated tissues and cells. We synthesized ET-1 labeled with I-123 and obtained images of receptor distribution in both rat and rhesus monkey and have demonstrated our ability to visualize, via planar, noninvasive imaging, the occupancy of endothelin receptor by antagonists in both kidney and lung. [123I]ET-1 can therefore be used to determine clinical dosages of antagonist needed for receptor saturation.

Risperidone: treatment response in adult and geriatric patients.

OBJECTIVE: To compare the efficacy and side effects of risperidone in younger adult and geriatric patients. METHODS: Open retrospective study of 102 consecutive intakes, prescribed risperidone, by a mental health team. All patients were non-hospitalized community residents. Prior to initiation of risperidone, and at termination of study period, Clinical Global Impression (CGI) scores were used to track progress. Variables monitored were: concurrent use of other antipsychotics, compliance, side effects, and maintenance dosage. RESULTS: The most common DSM-IV diagnoses were schizophrenia in the younger adult group and late onset delusional disorders in the geriatric group. Compliance was good for both groups. The geriatric group demonstrated a greater treatment response which was reached at a significantly lower dosage. There was no statistically significant difference in the occurrence of side effects. Examination of response by diagnostic category indicated that geriatric patients with late onset delusional disorder showed the best response while adults with either schizophrenia or affective syndromes also showed positive response. CONCLUSIONS: Risperidone, at lower than recommended doses, shows promise in the treatment of late onset delusional disorders and behavior syndrome of dementia. The side effect profile was benign, as was suggested by experience in treating schizophrenia. Scientifically more rigorous prospective studies for the indications and efficacy of risperidone in late onset psychotic disorders and psychoses and behavior syndromes associated with dementing illness are overdue.

A novel angiotensin II binding site in murine tissues.

A novel binding site for angiotensin II has been identified in certain murine tissues. This site, denoted ATm, is distinct from both the AT1 and AT2 sites, as well as the various atypical sites that have been described. The site has a low affinity for angiotensin II (100 nM) and is not blocked by losartan, PD123177 or saralasin. The site shows structural specificity for angiotensin II since both angiotensin III and angiotensin II (1-7) failed to inhibit the binding. Numerous standard drugs, including various receptor blockers, enzyme inhibitors and therapeutic agents, showed no affinity for the site. In murine tissues the site is associated with active tissue generation, as found in spleen, placenta and growing tumors, but its occurrence appears to be rare outside of the mouse.

Characterization of specific binding of [125I]L-762,459, a selective alpha1A-adrenoceptor radioligand to rat and human tissues.

L-762,459 ((+/-)1-(3-¿[5-carbamoyl-2-2-[(4-hydroxy-3-iodobenzimidoyl)-amino] -ethoxy-methy¿-6-methyl-4-(4-nitropheny)-1,4-dihydropyridine -3-carbonyl]-amino¿-propyl)-4-phenyl-1-piperidine-4-carboxylic acid methyl ester), an analog of a series of dihydropyridines previously reported to be selective alpha1A-adrenoceptor subtype antagonists was found to have alpha1A-adrenoceptor subtype selectivity (Ki (nM), la = 1.3, lb = 240, Id = 280). Specific [125I]L-762,459 binding was detected in rat cerebral cortex, hippocampus, vas deferens, kidney, heart and prostate tissues known to contain the alpha1A-adrenoceptor subtype, but not in tissues known to contain alpha1B-adrenoceptor (spleen, liver) and alpha1D-adrenoceptor (aorta). Scatchard analysis of [125I]L-762,459 binding in rat cerebral cortex and prostate indicated a single binding site with a Kd of 0.7 nM and Bmax of 11 (cerebral cortex) and 1 (prostate) pmole/g tissue. Specific and saturable [125I]L-762,459 binding was also found in human cerebral cortex, liver, prostate and vas deferens (Kd = 0.2-0.4 nM, Bmax = 0.4-4 pmole/g tissue). The specific binding in rat and human tissues was competed by non-selective alpha1-adrenoceptor compounds (Ki values in nM: prazosin (0.14-1.2), terazosin (1.8-5.9) and phentolamine (2.4-11)) and selective alpha1A-adrenoceptor compounds [Ki values in nM: (+) niguldipine (0.04-1.2) and SNAP 5399 ((+/-)-2-((2-aminoethyl)oxy)methyl-5-carboxamido-6-ethyl-4-(4-nitropheny l)-3-N-(3-(4,4-diphenylpiperidin-1-yl)propyl)carboxamido-1,4-dihyd ropyridine hydrate (0.5-4.8)]. The results were consistent with the selective binding of [125I]L-762,459 to the alpha1A-adrenoceptor. The specific labeling of the alpha1A-adrenoceptor subtype by [125I]L-762,459 may make it a useful tool to localize the distribution of the alpha1A-adrenoceptor.

Investigation of angiotensin II/AT1 receptors with carbon-11-L-159,884: a selective AT1 antagonist.

UNLABELLED: Antagonists of the angiotensin II AT1 receptor subtype have been recently introduced for treatment of arterial hypertension and for pharmacological studies of these receptors. The purpose of this work was to label such an antagonist with 11C and test the applicability of the radioligand for PET studies. METHODS: The potent and selective nonpeptide AT1 antagonist L-159,884 was labeled with 11C and injected intravenously into six dogs. Renal accumulation and kinetics of the radioligand were imaged with PET at baseline and after receptor blockade with 1 mg/kg MK-996. Time-activity curves were derived from the renal cortex and were analyzed by the Gjedde-Patlak plot to obtain the influx rate constant of the radioligand. RESULTS: There was selective radioligand binding in the kidneys, mainly located in the cortex. Within the time interval between 95 and 115 min postinjection, the radioactivity retained in the kidneys was 109 +/- 27 and 42 +/- 4 nCi/ml/mCi of the injected dose for the control and inhibition studies, respectively. The influx rate constant of the radioligand decreased from a baseline of 0.0298 +/- 0.0156 to a post-MK-996 value of 0.0098 +/- 0.0052. CONCLUSION: These results demonstrate distinct binding of 11C-L-159,884 in the renal cortex with a specific binding component suitable for quantitative PET imaging of angiotensin II/AT1 receptors.

Evaluation of a bicycle helmet giveaway program--Texas, 1995.

OBJECTIVE: To determine the effect of a bicycle helmet giveaway program on helmet use among children. METHODS: In 1995, a bicycle helmet giveaway program was conducted in two rural towns in Texas. Helmets were given to all 403 school children in kindergarten through grade 8. Helmet education, a bicycle rodeo, and incentives to increase helmet use were part of the program. Observations of helmet use were made before the helmet program began and after the program at several intervals throughout the school year and during the summer. A self-reported survey questionnaire was administered to children in grades 4 through 8 before the helmet program began and at several intervals during the school year to determine their attitudes about helmet use, safety perceptions, and peer pressure. A questionnaire also was administered to the parents of these children to determine attitudes and bicycle helmet use among parents. RESULTS: Helmet use increased from 3% before the giveaway to 38% at the end of the school year, 7 months later. However, during the subsequent summer, helmet use decreased to 5%. Helmet use among 7th- and 8th-grade students was 0% at all observations periods after the giveaway. Even though 96% of all students thought that helmet use increased riding safety and 68% thought helmets should be worn at all times when riding, only 25% thought that their friends would approve of helmet use. Most parents also believed that helmets increased riding safety and should be worn, but only 23% reported always wearing one when riding a bicycle. CONCLUSIONS: Bicycle helmet giveaway programs can increase helmet use temporarily, but they may not be sufficient to sustain it. This program was not effective among 7th- and 8th-grade students.

Opsonin-independent adherence and phagocytosis of Listeria monocytogenes by murine peritoneal macrophages.

Listeria monocytogenes adhered to and multiplied intracellularly in murine peritoneal macrophages in the absence of opsonins. The infective process in these cells was evaluated by viable bacterial cell colony counts of intracellular organisms and documented by transmission and scanning electron microscopy. Adherence of listeriae to macrophages involved surface interactions of the prokaryotic cell surface and eukaryotic cell membranes. Subsequent phagocytosis was seen to occur through a process in which host cell-derived pseudopodia surrounded and engulfed organisms leaving them within phagosomes in the cytoplasm of infected cells. This process of uptake of L. monocytogenes by macrophages occurred at 4 degrees C. Following invasion of the cell, escape of L. monocytogenes from the phagosome into the cytoplasm was initiated as early as 10 min into the infective process. Intracellular multiplication of bacteria continued for 8 h after inoculation at which point loss of adherent macrophages due to cell lysis was evident. The mean generation time of the organism in these cells was 58 min. The cellular and ultrastructural events of L. monocytogenes adherence to and phagocytosis by murine macrophages in the absence of antibody or complement have been defined.

In vivo labeling of angiotensin II receptors with a carbon-11-labeled selective nonpeptide antagonist.

UNLABELLED: Angiotensin II (ANG II) initiates a variety of physiological effects by binding to high affinity receptors. Two ANG II receptor subtypes, AT1 and AT2, have recently been identified. This study was undertaken to evaluate [11C]L-159,884, an AT1 subtype selective nonpeptide antagonist, as a potential PET tracer. METHODS: Carbon-11-L-159,884 was prepared by alkylation of the nor precursor with [11C]methyliodide and was studied for its in vivo binding characteristics, biodistribution and kinetics in mice. The effects of PD-123319, an AT2-selective ANGII antagonist, as well as those of alpha- and beta-adrenergic drugs on [11C]L-159,884 binding were investigated also. RESULTS: Administration of the AT1 antagonists resulted in dose-dependent inhibition of [11C]L-159,884 binding in the kidneys, the organ with the highest density of AT1 receptors. Inhibition was also observed in the lungs and the heart. Adrenergic drugs did not influence [11C]L-159,884 binding to AT1 receptors. Kinetic studies showed rapid tracer uptake in the liver, kidneys, lungs and heart. Excretion of the radioactivity occurred primarily through the intestinal tract (> 20% in 90 min), with less than 8% excreted through the urine. CONCLUSION: The results suggest that [11C]L-159,884 binds in vivo to AT1 receptors in mouse kidneys, lungs and heart. This radiotracer appears to be a promising candidate for studying ANG II receptors in vivo by PET.

Nonpeptide angiotensin II receptor antagonists: in vivo inhibition of [125I-Sar1,Ile8]angiotensin II binding by losartan, EXP597 and L-159,282 in rats.

Effects of losartan, L-159,282 and EXP597 on the in vivo binding of [125I-Sar1,Ile8]angiotensin II to kidney cortex and adrenal were examined in rats. Losartan, an AT1 receptor antagonist, completely blocked [125I-Sar1,Ile8]angiotensin II binding to the kidney cortex which contains only AT1 binding sites with an ID50 of 0.06 mg/kg. Losartan partially inhibited [125I-Sar1,Ile8]angiotensin II binding to the adrenal which contains equal amounts of AT1 and AT2 binding sites. Blockade by the AT1 receptor antagonist L-159,282 sufficiently increased the plasma levels of angiotensin II to block the AT2 receptor. EXP597 inhibited [125I-Sar1,Ile8]angiotensin II binding to the kidney cortex and adrenal almost totally with ID50s of 0.05 and 0.06 mg/kg, respectively. This result suggests that EXP597 exhibits almost equal binding affinity for AT1 and AT2 binding sites in vivo in rats.

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors.

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.

Autoradiographic evidence that QNB displays in vivo selectivity for the m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. We have previously reported the results of in vivo dissection studies, using both carrier-free and low specific activity [3H]QNB, which show that [3H]QNB exhibits a substantial in vivo m2 selectivity. Because of the expense of the radioligand and the long exposure time required for the X-ray film, performing a large number of direct in vivo autoradiographic studies using [3H]QNB is precluded. Therefore, we now confirm these results autoradiographically by studying the in vivo inhibition of radio-iodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled QNB. In the absence of QNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 15 nmol QNB, (R,S,)-[125I]IQNB labeling in those brain regions containing predominantly m2 subtype is reduced to background levels. We conclude that QNB is m2-selective in vivo and that a suitably radiolabeled derivative of QNB, possibly labeled with 18F, may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD.

In vivo receptor occupancy of the angiotensin II receptor by nonpeptide antagonists: relationship to in vitro affinities and in vivo pharmacologic potency.

The affinities of 13 angiotensin II antagonists for the AT1 subtype determined in vitro with tissue homogenates were shown not to correlate well with in vivo pharmacologic potency. The addition of human serum albumin to the in vitro assay to mimic in vivo plasma protein interactions reduced the measured affinity by reducing the effective free concentrations of antagonists, but the resulting affinities were not predictive of the in vivo effects. Using an in vivo radioligand competition assay, in which receptor occupancy is demonstrated via competitive blockade of the in vivo binding of [125I][Sar1,Ile8]angiotensin II to AT1 receptors in rat kidney cortex, we demonstrated that the in vivo pharmacologic potencies reflect receptor occupancy. By comparing the effects of rat plasma and bovine serum albumin on the in vitro affinity of two antagonists, we suggest that the use of plasma would alter free plasma concentrations in a manner more consistent with in vivo measures of potencies.

Characterization of the binding of [125I]L-735,286: a new nonpeptide angiotensin II AT1 receptor radioligand.

[125I]L-735,286, a new potent and AT1-selective nonpeptide angiotensin II receptor radioligand, bound saturably to whole adrenal membranes. Scatchard and Hill plot analysis indicates a single class of high affinity (Kd = 0.5 nM) binding sites. The potencies of various angiotensin II agonists and antagonists in displacing specific [125I]L-735,286 binding are in good agreement with their potencies in displacing the binding of [125I]Sar1,Ile8-AII to adrenal AT1 receptors. The AT2 selective ligand, PD121981 had no effect on specific [125I]L-735,286 binding. In autoradiographic studies using rat kidney slices, specific labeling of [125I]L-735,286 was abolished by coincubation with saralasin. Collectively, the data indicated that [125I]L-735,286 represents a new, potent nonpeptide antagonist radioligand suitable for the study of angiotensin II AT1 receptors.

Receptor binding radiotracers for the angiotensin II receptor: radioiodinated [Sar1, Ile8]Angiotensin II.

The potential for imaging the angiotensin II receptor was evaluated using the radioiodinated peptide antagonist [125I][Sar1, Ile8)angiotensin II. The radioligand provides a receptor-mediated signal in several tissues in rat (kidneys, adrenal and liver). The receptor-mediated signal of 3% ID/g kidney cortex should be sufficient to permit imaging, at least via SPECT. The radiotracer is sensitive to reductions in receptor concentration and can be used to define in vivo dose-occupancy curves of angiotensin II receptor ligands. Receptor-mediated images of [123I][Sar1, Ile8]angiotensin II were obtained in the rat kidney and Rhesus monkey liver.

Muscarinic receptor selectivities of 3-Quinuclidinyl 8-xanthenecarboxylate (QNX) in rat brain.

We have determined the binding of (R)-3-Quinuclidinyl 8-xanthenecarboxylate to muscarinic acetylcholine receptor preparations from rat cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The competition curves determined with [3H]quinuclidinyl benzilate as the radioligand are well described by a two site model with a difference in affinity between the two sites of 12-fold. The proportions of high affinity site vary from 100% in the caudate/putamen to 0% in the pons/medulla. The selectivities are different from those measured by pirenzepine and are consistent with QNX exhibiting similar affinity for the M1, M3, and M4 receptors with lower affinity for the M2 receptor. This assignment was confirmed by determining the affinities of QNX for the cloned receptor subtypes.

Antithrombotic activity of recombinant tick anticoagulant peptide and heparin in a rabbit model of venous thrombosis.

An in vivo rabbit model of venous thrombosis which includes physiological blood flow was used to compare the efficacy of the potent and specific factor Xa inhibitor recombinant tick anticoagulant peptide (rTAP) with standard heparin in the prevention of venous thrombus formation. In anesthetized rabbits, an autologous thrombus was induced with thrombin in a jugular vein and the increase in thrombus size was determined by measuring the accretion of intravenously injected [125I]fibrin(ogen) onto the developing thrombus. The effects of rTAP on hemostasis were monitored by changes in APTT values and template bleeding times. Inhibition of thrombus formation by an intravenous bolus followed by infusion of either rTAP or heparin exhibited a dose-response relationship with an IC50 of 0.9 micrograms/kg/min and 0.12 units/kg/min, respectively. At the IC50 doses, both rTAP and heparin inhibited fibrin(ogen) deposition without any significant effect on APTT or bleeding times. Bleeding times were modestly elevated at the fully efficacious doses of rTAP and heparin. Significant changes in APTT (1.9 +/- 0.3 fold over baseline) were only evident at the highest dose of rTAP while heparin caused a significant dose-dependent increase from 1.3 +/- 0.2 to greater than 4.2 +/- 0.6 fold over baseline. Therefore, in this rabbit model of venous thrombosis, specific inhibition of factor Xa by rTAP is an effective antithrombotic mechanism that does not require changes in systemic hemostatic parameters.