Non-mammalian fat-1 gene prevents neoplasia when introduced to a mouse hepatocarcinogenesis model: Omega-3 fatty acids prevent liver neoplasia.

We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-alpha (transforming growth factor-alpha), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFalpha/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFalpha/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-kappaB (nuclear factor-kappaB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.

Relationships between growth and disease resistance in rainbow trout, Oncorhynchus mykiss (Walbaum).

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post-fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.

Magnetic resonance spectroscopy for evaluation of liposome-encapsulated hemoglobin as a resuscitation fluid.

Liposome-encapsulated hemoglobin (LEH) based on a novel, synthetic, non-phospholipid was developed, and evaluated for cerebral energy metabolism in a 40% hemorrhage rat model. The markers of tissue energetics were monitored by (1)H- and (31)P-magnetic resonance spectroscopy (MRS). After hemorrhage, (1)H-MRS showed an increase in the levels of lactate and pyruvate. These markers returned to baseline values following LEH resuscitation. Both LEH and saline were able to exert a neuron-protective effect as indicated by the recovery of N-acetylaspartate. (31)P MRS showed a fall in phosphocreatine after hemorrhage, which upon LEH or saline resuscitation returned to the baseline values. Similarly, inorganic phosphate increased after bleeding, but returned to normal after resuscitation. LEH resuscitation also recovered beta-ATP levels, but saline resuscitation provided only a modest recovery. The results indicate the utility of MRS to monitor cerebral metabolism in hemorrhage/resuscitation. The data is also supportive of the new LEH formulation as an oxygen carrier.

Application of proton NMR spectroscopy in the study of lipid metabolites in a rat hepatocarcinogenesis model.

Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver is a key feature of developing liver nodules and tumors. Methods of analysis vary from the most sophisticated chromatography to the in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, we present a systematic method for the identification and quantitation of signature signals from lipid metabolites using 1D NMR proton spectroscopy. We assessed lipid metabolites in an epigenetic rat hepatocarcinogenesis model induced by treatment with a choline-deficient diet (CDAA, choline-deficient l-amino acid defined) over a period of 1 year, from the formation of steatosis, to the development of nodules and adenomas. A comparable choline-sufficient (CSAA) diet was used for the controls. The resonances of the methylene protons of the glycerol backbone in phospholipids were used to quantify the total concentration of such compounds. CDAA rat livers were found to have significantly higher levels of phospholipids, when compared to CSAA, throughout the entire carcinogenesis period. The tri-methyl protons of choline compounds serves to quantify total choline, and the vinyl and bis-allyl proton resonances can be used to not only quantify fatty acid concentrations but also to probe the number of double bonds in a fatty acid moiety. Early stages of carcinogenesis indicate a lower degree of double bonds in fatty acyl containing compounds in CDAA rat livers, when compared to CSAA. The results of this study are in agreement with those previously published in the literature on other rat hepatocarcinogenesis models.

In vivo assessment of microcystin-LR-induced hepatotoxicity in the rat using proton nuclear magnetic resonance (1H-NMR) imaging.

Microcystin-LR (MCLR)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-350 g) using magnetic resonance imaging (MRI). Following the intraperitoneal administration of MCLR (LD(50)), a region of damage, characterised by increased signal intensity on T(2)-weighted images, was seen proximal to the hepatic portal vein in the liver. Similarly, increased signal intensity was seen in the chemical-shift selective images (CSSI) of water frequency, proximal to the hepatic portal vein in the liver. This indicates that the increased signal intensity observed in the T(2)-weighted images was due to an increased amount of magnetic resonance (MR) visible protons in the tissue which represents an oedematous response. Image analysis of regions of apparent damage around the hepatic portal vein indicated a statistically significant increase in signal intensity in this region. Mitochondrial swelling and lipid inclusions were observed by transmission electron microscopy (TEM) in samples obtained from the oedematous regions of the liver using spatial coordinates from the magnetic resonance (MR) images. Massive haemorrhagic necrosis and nuclear swelling were observed by light microscopy in the centrilobular regions of the lobules.

Non-invasive in vivo magnetic resonance imaging assessment of acute aflatoxin B1 hepatotoxicity in rats.

Acute aflatoxin B1 (AFB1)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-300 g) using magnetic resonance imaging (MRI). MRI results were compared to serum enzyme levels, histology and electron microscopy. Twenty-four hours following intraperitoneal delivery of AFB1 (3 mg/kg body weight in a saline/dimethyl sulfoxide (DMSO; 0.03 ml/kg body weight) solution), regions of damage, characterised by increased proton signal intensities in T2-weighted images, were observed in the vicinity of the hepatic portal vein (HPV) and in the right medial regions of the liver. Image analysis of regions of apparent damage around the HPV and right medial regions, following 24 h of AFB1 exposure, indicated statistically significant (P<0.05) increases in proton image signal intensities, when compared to saline/DMSO-treated rats. No significant difference in proton image signal intensities were observed 1-2 h following AFB1 exposure. Twenty-four hours following AFB1 exposure, histopathological assessment was characterised by portal/central vein/artery congestion, sinusoid congestion, nuclear pyknosis and karyolysis, and hepatocyte vacuolation; electron microscopy (EM) examination indicated nuclear debris, swollen cytoplasmic compartments, vacuolation, and the disappearance of the smooth endoplasmic reticulum, and elevated levels of serum aspartate aminotransferase and alanine aminotransferase were found to be significantly different (P<0.01) than controls.

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons.

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.

Enhancement of carbon tetrachloride-induced liver injury by a single dose of ethanol: proton magnetic resonance imaging (MRI) studies in vivo.

Magnetic resonance imaging (MRI) and localized magnetic resonance spectroscopy (MRS) were used to study the effects of a single dose of ethanol, given 18 h prior to experiments, on CC14-induced acute hepatotoxicity in rats in situ. Localized edema in the centrilobular region of the liver, following exposure to ethanol and CCl4, was detected by 1H-MRI techniques. The edema was characterized by a volume selective spectroscopy (VOSY) method, which measured an increase in water concentration from ethanol and CCl4-treated rat livers, in comparison to control livers. Electron microscopy (EM) of the high intensity regions of the ethanol/CCl4 treated liver sections revealed dramatic subcellular changes such as fragmentation of the granular endoplasmic reticulum (ER), formation of large vacuoles and lipid droplets in the cytoplasmic matrix and extensive swelling of the mitochondria as well as disruption of the cristae. Pretreatment with alpha-phenyl tert-butyl nitrone (PBN), a free radical spin trap, prior to halocarbon exposure, was found to reduce the CC14-mediated high intensity region in the liver images. Electron microscopy of the PBN pretreated CCl4 exposed rat liver sections revealed only minor observable differences in subcellular organization, such as some swelling of the mitochondria, when compared to controls. In addition, these data suggest that ethanol may potentiate CCl4 hepatotoxicity by increased formation of free radical intermediates. Inhibition of the CCl4-induced edematous response in rat liver by PBN demonstrates that free radical intermediates, arising from the metabolism of CCl4, are possibly the causal factor in the initiation of the edema.

In vivo image-guided (1)H-magnetic resonance spectroscopy of the serial development of hepatocarcinogenesis in an experimental animal model.

Histology on a core or open biopsy is considered the gold standard for the diagnosis of tumours. While the non-invasive technique of magnetic resonance imaging can direct some of the decision diagnostic making, it has limitations and disadvantages, that can be partly overcome with the use of in vivo magnetic resonance spectroscopy (MRS). In vivo MRS is able to provide a specific biochemical profile on tumour tissue, compared with normal tissue. The capability of this technique is demonstrated here by the long-term development of hepatocellular carcinoma in an animal model. It allows the observation of the biochemical changes that occur in tumour tissue during its progression from preneoplastic nodules to hepatocellular carcinoma. Specifically the changes in the lipid profiles of tumour tissue at various stages of development are observed with proton ((1)H) MRS. Significant increases occurred in the lipid acyl chain methylene and methyl hydrogens during the early developmental stages of hepatocarcinogenesis, whereas during later stages associated with tumour development there was a significant increase in the levels of olefinic acyl chain hydrogens from unsaturated lipids. It is anticipated that this model will precede the application of the same technology to the non-invasive diagnosis and grading of human hepatocellular carcinoma.

In vivo detection of aflatoxin-induced lipid free radicals in rat bile.

Aflatoxin B1 (AFB1), a potent hepatotoxin and hepatocarcinogen, is metabolized in the liver via cytochrome P-450 to an AFB1-8,9-epoxide intermediate. The formation of the AFB1-8,9-epoxide correlates with the pathological changes observed in numerous mammalian species. Oxidative damage has been postulated to play a major role in the mechanisms associated with AFB1-induced cytotoxicity and carcinogenecity in mammalian species. The aim of this study was to detect and identify free radical intermediates from the hepatic metabolism of AFB1 in vivo. Rat bile ducts were cannulated and rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone) (1 g/kg i.p.), and bile was collected over a period of 2 h at 20-min intervals. ESR spectroscopy was used to detect a carbon-centered radical adduct of 4-POBN in rat bile. The effect of metabolic inhibitors, such as deferoxamine mesylate (DFO), an iron chelator, and SKF 525A, a cytochrome P-450 inhibitor, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in free radical formation by pre-treatment with both DFO and SKF 525A. This indicates that oxidation of AFB1 generates free radical species via CYP metabolism and an iron-mediated redox mechanism.

Hydroxyl radical generation following ischaemia-reperfusion in cell-free perfused rat kidney.

The difficulty in direct detection of oxygen-derived free radicals (OFR) in the intact kidney has left uncertain the role of OFR in renal hypoperfusion injury. Salicylate hydroxylation was used as a sensitive method of estimating the extent of production of highly reactive hydroxyl radicals in renal ischaemia-reperfusion injury in the intact rat kidney perfused with recirculating cell-free medium. The reaction products were detected and quantified by HPLC with electrochemical detection. Hydroxyl radicals were detected as 2,5-dihydroxybenzoic acid (2,5-DHBA). Ischaemia for 15 min followed by reperfusion for 15 min caused more than a twofold increase in 2,5-DHBA concentration (to 2279 +/- 225 pg/g tissue weight) compared to controls (933 +/- 103, P < 0.001). Addition of 15 mM dimethylthiourea (DMTU) before induction of ischaemia prevented this increase. Induction of hypoxia for 15 min with continued perfusion (as a model of low-flow ischaemia) had no significant effect on hydroxyl radical formation. We conclude that significant quantities of hydroxyl radicals form in the absence of circulating leucocytes during reperfusion following ischaemia, but not during hypoxia in the perfused rat kidney.

In vivo targeted molecular magnetic resonance imaging of free radicals in diabetic cardiomyopathy within mice.

Free radicals contribute to the pathogenesis of diabetic cardiomyopathy. We present a method for in vivo observation of free radical events within murine diabetic cardiomyopathy. This study reports on in vivo imaging of protein/lipid radicals using molecular MRI (mMRI) and immuno-spin trapping (IST) in diabetic cardiac muscle. To detect free radicals in diabetic cardiomyopathy, streptozotocin (STZ)-exposed mice were given 5,5-dimethyl-pyrroline-N-oxide (DMPO) and administered an anti-DMPO probe (biotin-anti-DMPO antibody-albumin-Gd-DTPA). For controls, non-diabetic mice were given DMPO (non-disease control), and administered an anti-DMPO probe; or diabetic mice were given DMPO but administered a non-specific IgG contrast agent instead of the anti-DMPO probe. DMPO administration started at 7 weeks following STZ treatment for 5 days, and the anti-DMPO probe was administered at 8 weeks for MRI detection. MRI was used to detect a significant increase (p < 0.001) in MRI signal intensity (SI) from anti-DMPO nitrone adducts in diabetic murine left-ventricular (LV) cardiac tissue, compared to controls. Regional increases in MR SI in the LV were found in the apical and upper-left areas (p < 0.01 for both), compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised cardiac tissues, which indicated elevated fluorescence only in cardiac muscle of mice administered the anti-DMPO probe. Oxidized lipids and proteins were also found to be significantly elevated (p < 0.05 for both) in diabetic cardiac muscle compared to controls. It can be concluded that diabetic mice have more heterogeneously distributed radicals in cardiac tissue than non-diabetic mice.

Assessment of colon and bladder crosstalk in an experimental colitis model using contrast-enhanced magnetic resonance imaging.

BACKGROUND: Inflammatory bowel disease (IBD) consists of two chronic remitting-relapsing inflammatory disorders in the colon referred to as ulcerative colitis and Crohn's disease (CD). Inflammatory bowel disease affects about 1.4 million Americans. 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis is a widely used model of experimental intestinal inflammation with characteristic transmural and segmental lesions that are similar to CD. METHODS: Here, we report on the use of contrast-enhanced magnetic resonance imaging (CE-MRI) to monitor in vivo bladder permeability changes resulting from bladder crosstalk following colon TNBS exposure, and TNBS-induced colitis. Changes in MRI signal intensities and histology were evaluated for both colon and bladder regions. KEY RESULTS: Uptake of contrast agent in the colon demonstrated a significant increase in signal intensity (SI) for TNBS-exposed rats (p < 0.01) compared to controls. In addition, a significant increase in bladder SI for colon TNBS-exposed rats (p < 0.001) was observed compared to saline controls. Histological damage within the colon was observed, however, bladder histology indicated a normal urothelium in rats with TNBS-induced colitis, despite increased permeability seen by CE-MRI. CONCLUSIONS & INFERENCES: Contrast-enhanced MRI was able to quantitatively measure inflammation associated with TNBS-induced colitis, and assess bladder crosstalk measured as an increase in urothelial permeability. Although CE-MRI is routinely used to assess inflammation with IBD, currently there is no diagnostic test to assess bladder crosstalk with this disease, and our developed method may be useful in providing crosstalk information between organ and tissue systems in IBD patients, in addition to colitis.

MRI study of the inhibitory effect of new spin traps on in vivo CCl4-induced hepatotoxicity in rats.

Acute CCl4 hepatotoxicity is thought to occur as a result of free radicals generated from the metabolism of CCl4 in the liver. With the use of MRI it is possible to detect in vivo a CCl4-induced localized edematous region surrounding the major branch of the hepatic portal vein in the right lobe. Inhibition of the CCl4-induced response has been obtained by pretreatment with the spin trap, PBN, 30 min prior to CCl4 exposure. The inhibitory effect of two new spin traps, M3PO or methyl-DMPO, and PhM2PO or phenyl-DMPO, on in vivo CCl4-induced acute hepatotoxicity was investigated. Both PhM2PO and M3PO were found to inhibit the CCl4-induced response at lower concentrations (0.35 M/kg body weight) than PBN (0.70 M/kg body weight). However, both M3PO and PhM2PO were also found to induce an edematous response at the same concentrations used for the PBN studies (0.70 M/kg body weight). PhM2PO, at a concentration of 0.35 M/kg body weight, was 93% as efficient as PBN, at a concentration of 0.70 M/kg body weight; whereas M3PO, at a concentration of 0.35 M/kg, was 89% as efficient as PBN at 0.70 M/kg body weight. Electron micrographs were obtained from small liver sections taken in proximity to the major branch of the hepatic portal veins of all treatment groups. The electron microscopy investigations support the MRI findings.

MRI detection of hyperoxia-induced lung edema in Zn-deficient rats.

In a pioneering application of proton Magnetic Resonance Imaging (MRI), lung edema has been monitored in vivo in Zn-deficient rats exposed to 85% oxygen. Dietary Zn appears to play a role in protecting against hyperoxia-induced lung damage.

Phenothiazines as lipid peroxidation inhibitors and cytoprotective agents.

A series of phenothiazines was synthesized and evaluated as in vitro inhibitors of iron-dependent lipid peroxidation. The MIC (minimum tested concentration that gave greater than or equal to 50% inhibition) for 2-(10H-phenothiazin-2-yloxy)-N,N-dimethylethanolamine methanesulfonate (6) was 0.26 microM. Whereas methyl substitution at N-10 diminished activity nearly 100-fold, other structural modifications such as varying the amine group, the distance separating the amine substituent from the phenothiazine nucleus, and the linking group had little effect. Compound 6 was more effective than probucol, a known antioxidant, in blocking Cu2+ catalyzed oxidation of low-density lipoprotein (LDL) as measured by competitive scavenger receptor mediated degradation of 125I-labeled acetyl-LDL by mouse peritoneal macrophage cells in vitro. At a concentration of 5 microM, compound 6 also protected primary cultures of rat hippocampal neurons exposed to hydrogen peroxide (50 microM) when assessed 18 h later by fluorescein diacetate and propidium iodide uptake.

Benzylamine antioxidants: relationship between structure, peroxyl radical scavenging, lipid peroxidation inhibition, and cytoprotection.

Three homologous series of 3,5-dialkoxy-4-hydroxybenzylamines were prepared and tested (1) as peroxyl radical scavengers in homogeneous aqueous solution, (2) as inhibitors of iron-dependent peroxidation of rabbit brain vesicular membrane lipids, and (3) as cytoprotective agents using primary cultures of rat hippocampal neurons exposed to hydrogen peroxide. The structural requirements for efficient radical trapping in homogeneous solution differed from those for effective lipid peroxidation inhibition: In homogeneous solution a kinetic preference existed for smaller, less sterically encumbered substituents flanking the reactive phenolic hydroxyl group. Lipid peroxidation inhibition, on the other hand, required longer more lipophilic substituents. Consequently, a lipophilic alkoxyl substituent at C3 and a small substituent at C5 appeared optimal for efficient radical scavenging activity in both lipid and homogeneous solution. Maximal cytoprotection of rat hippocampal neurons exposed to hydrogen peroxide was also associated with more lipophilic derivatives although substituent length and substituent bulk may represent independent parameters for relating structure and efficacy in this system.

N(2)-Aroylanthranilamide inhibitors of human factor Xa.

Reversal of the A-ring amide link in 1,2-dibenzamidobenzene 1 (fXa K(ass) = 0.81 x 10(6) L/mol) led to a series of human factor Xa (hfXa) inhibitors based on N(2)-aroylanthranilamide 4. Expansion of the SAR around 4 showed that only small planar substituents could be accommodated in the A-ring for binding to the S1 site of hfXa. Bulky groups such as 4-isopropyl, 4-tert-butyl, and 4-dimethylamino were favored in the B-ring to interact with the S4 site of hfXa. The central (C) ring containing a 5-methanesulfonamido group yielded greater activity than carbamoyl groups. Combining the beneficial features from the B- and C-ring SAR, compound 55 represents the most potent hfXa inhibitor in the N(2)-aroylanthranilamide 4 series with hfXa K(ass) = 58 x 10(6) L/mol (K(i) = 11.5 nM).

Fused bicyclic Gly-Asp beta-turn mimics with specific affinity for GPIIb-IIIa.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6, 5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

1,2-Dibenzamidobenzene inhibitors of human factor Xa.

High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound 4, fXa apparent K(ass) = 0.64 x 10(6) L/mol). SAR studies in this novel structural series of fXa inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tert-butyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced slightly higher levels of activity than other substituents when present in combination with favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds, having fXa apparent K(ass) = 25.5 x 10(6) L/mol, about 40x more active than the original lead. This lead series does not show potent inhibition of human thrombin. A model for the binding of these ligands to the fXa active site is proposed. The model is consistent with the observed SAR and can serve to guide future SAR studies.