Modulation of nutrient sensing nuclear hormone receptors promotes weight loss through appetite suppression in mice.

AIM: Peroxisome proliferator activated receptors (PPARs) are nuclear receptors involved in glucose and lipid metabolism. Three isoforms of PPARs have been identified with different tissue distribution and biological functions. Although the pharmacology of each receptor is well studied, the physiological effect of simultaneous activation of PPARalpha, gamma and delta is only starting to emerge. We sought to determine the biological effects of a novel PPAR pan activator and elucidate the physiological mechanisms involved. METHODS: Ob/ob, diet-induced obese (DIO) or PPARalpha knockout mice were administered a novel agonist that activates all PPARs to various degrees to determine the effect on body weight, body composition, food intake and energy expenditure. In addition, serum parameters including glucose, insulin, triglycerides and ketone bodies as well as tissue acylcarnitine were evaluated. The effect of the novel agonist on liver and skeletal muscle histopathology was also studied. RESULTS: We report that simultaneous activation of all PPARs resulted in substantial weight loss in ob/ob and DIO mice. Consistent with known PPAR pharmacology, we observed that agonist treatment increased lipid oxidation, although appetite suppression was mainly responsible for the weight loss. Agonist-induced weight loss was completely absent in PPARalpha knockout mice suggesting that PPARalpha pharmacology was the major contributor to weight regulation in mice. CONCLUSIONS: Our work provides evidence that simultaneous activation of PPARalpha, gamma and delta decreases body weight by regulating appetite. These effects of the pan agonist were completely absent in PPARalpha knockout mice, suggesting that PPARalpha pharmacology was the major contributor to weight loss.

Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa.

A low molecular weight serine protease inhibitor (TAP) was purified from extracts of the soft tick, Ornithodoros moubata. The peptide is a slow, tight-binding inhibitor, specific for factor Xa (Ki = 0.588 +/- 0.054 nM). The inhibitor also acts as an anticoagulant in several human plasma clotting assays in vitro. Its amino acid sequence (60 residues) has limited homology to the Kunitz-type inhibitors. However, unlike other inhibitors of this class, TAP inhibits only factor Xa. It had no effect at a 300-fold molar excess on factor VIIa, kallikrein, trypsin, chymotrypsin, thrombin, urokinase, plasmin, tissue plasminogen activator, elastase, or Staphylococcus aureus V8 protease. TAP's specificity and size suggest that it may have therapeutic value as an anticoagulant.

Ability of recombinant factor VIIa to generate thrombin during inhibition of tissue factor in human subjects.

BACKGROUND: In view of the central role of the tissue factor-factor VIIa pathway in the initiation of blood coagulation, novel therapeutic strategies aimed at inhibiting this catalytic complex are currently being evaluated. A limitation of this new class of anticoagulants may be the lack of an appropriate strategy to reverse the effect if a bleeding event occurs. The aim of this study was to investigate the in vivo potential of recombinant factor VIIa (rVIIa) to induce thrombin generation in healthy subjects pretreated with recombinant nematode anticoagulant protein c2, a specific inhibitor of the tissue factor-factor VIIa complex, in a double-blind randomized crossover study. METHODS AND RESULTS: Administration of nematode anticoagulant protein c2 (3.5 microgram/kg) caused a prolongation of the prothrombin time from 13.7+/-0.6 to 16.9+/-1.2 seconds. The subsequent injection of rVIIa (90 microgram/kg) resulted in an immediate and complete correction of the prothrombin time and a marked generation of thrombin, reflected by increased levels of prothrombin activation fragment F1+2 and thrombin-antithrombin complexes from 0.75+/-0.64 to 3.29+/-6.3 nmol/L and from 2.4+/-0.6 to 10.7+/-3.9 microgram/mL, respectively. Factor X and IX activation peptides showed a 3.5-fold and a 3.8-fold increase, respectively, after the administration of rVIIa in the presence of nematode anticoagulant protein c2. CONCLUSIONS: During treatment with an inhibitor of the tissue factor-factor VIIa complex, the infusion of rVIIa resulted in thrombin generation. Our results indicate that rVIIa may be a good candidate as an antidote for inhibitors of tissue factor.

The role of CD11/CD18 integrins in the reverse passive Arthus reaction in rat dermal tissue.

The CD11/CD18 leukocyte integrins are necessary for tissue localization of neutrophils, an early requisite event in inflammation. We have analyzed the contribution of CD11a/CD18 and CD11b/CD18 to local neutrophil accumulation and tissue injury in the reverse passive Arthus reaction in the rat dermis. Experimental groups comprised animals that received an intravenous infusion of (1) recombinant neutrophil inhibitory factor (NIF), a hookworm-derived antagonist of CD11b/CD18; (2) monoclonal antibody to CD11a/CD18 (TA-3); (3) a combination of these agents; (4) a monoclonal antibody to CD18 (WT.3); or (5) saline. Administration of recombinant NIF or anti-CD11a/CD18 monoclonal antibody alone produced a slight reduction in neutrophil accumulation but did not affect edema formation. In contrast, a combination of these antagonists yielded a significant reduction in neutrophil accumulation and a modest reduction in edema, equivalent to levels observed with either anti-CD18 antibodies or animals that were rendered neutropenic. These results indicate that neutrophil infiltration in rat dermal tissue in the reverse passive Arthus reaction is dependent predominantly on the leukocyte integrins CD11a/CD18 and CD11b/CD18 and that either of these integrins is sufficient for neutrophil trafficking in this inflammatory setting.

Maintenance of canine coronary artery patency following thrombolysis with front loaded plus low dose maintenance conjunctive therapy. A comparison of factor Xa versus thrombin inhibition.

OBJECTIVE: The aim was to examine the abilities of the direct thrombin inhibitor, recombinant hirudin (rHIR), and the coagulation factor Xa inhibitor, recombinant tick anticoagulant peptide (rTAP), given in combination with rt-PA as high dose front loading plus low dose maintenance infusions, to enhance reperfusion and maintain vessel patency in a canine model of left circumflex coronary artery stenosis and electrolytic lesion. METHODS: Occlusive coronary artery thrombosis was induced in anaesthetised dogs by electrical injury (150 microA) of the intimal surface of the vessel. Thirty minutes after occlusive thrombosis, high dose front loading infusions (45 min) of rTAP (200 micrograms.kg-1 x min-1) and rHIR (300 micrograms.kg-1 x min-1) were initiated concomitant with the start of a 90 min infusion of recombinant tissue-type plasminogen activator (rt-PA). Following the termination of front loading infusions, maintenance infusions of rTAP (10 or 20 micrograms.kg-1 x min-1) or rHIR (20 micrograms.kg-1 x min-1) were initiated and continued for the duration of the protocol (180 min after rt-PA termination). RESULTS: Reperfusion was incomplete in the rHIR group (7/9; 78%), whereas all rTAP-treated preparations reperfused (8/8 per group, aggregate 16/16; 100%). Following thrombolysis, the rHIR group had a high incidence of reocclusion, ranging from intermittent to long periods of occlusion, with only 2/7 (29%) of the preparations which initially recanalised remaining patent during the 180 min period following rt-PA termination. In contrast, 5/8 preparations in each of the two rTAP groups [aggregate 10/16; 63%] remained patent during the same period. The greater efficacy of rTAP v rHIR in maintaining vessel patency was also reflected in integrated coronary artery blood flows [91.0(SEM 5.8)% and 84.9(6.1)% of preocclusion flow in rTAP groups v 57.5(12.2)% of preocclusion flow in rHIR group], times to reocclusion [123.3(22.8) and 128.0(6.7) min in rTAP groups v 36.6(23.2) min in rHIR group; p < 0.05], and residual thrombus masses [1.8(0.3) and 2.0(0.3) mg in rTAP groups v 10.4(3.8) mg in rHIR group; p < 0.05]. CONCLUSIONS: With the present front loading plus low dose maintenance infusions designed to limit the duration of "high dose" conjunctive therapy, rTAP was more effective than rHIR at equimolar plasma concentrations in maintaining post-thrombolysis vessel patency, preserving coronary artery blood flow, and reducing residual thrombus mass. These findings further support the therapeutic potential of inhibiting factor Xa in the setting of coronary artery thrombolysis.

NMR structure determination of tick anticoagulant peptide (TAP).

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.

Synthesis, structure, and structure-activity relationships of divalent thrombin inhibitors containing an alpha-keto-amide transition-state mimetic.

A new class of divalent thrombin inhibitors is described that contains an alpha-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the alpha-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their alpha-keto-amide counterparts. The most potent alpha-keto-amide inhibitor, CVS995, with a Ki = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.

Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats.

Intravenous (bolus) administration of endothelin results in a transient fall in blood pressure that is accentuated in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto normotensive rats (WKY). In attempting to discern possible mechanisms underlying this depressor response, we examined the ability of endothelin to release atrial natriuretic factor (ANF) from isolated, spontaneously contracting atria from SHR and WKY. Isolated right atria were suspended under 3.0 g of resting force in tissue baths with the amount of immunoreactive ANF (irANF) released after exposure to endothelin assessed by radioimmunoassay. Endothelin (10(-8) and 10(-7) M) caused a concentration-dependent increase (1.5-4.5-fold) in the release of irANF, which was significantly greater in atria of SHR compared with WKY. The greater release of irANF in atria of SHR versus WKY was not related to tissue weight or changes in contractile rate or force induced by endothelin. Therefore, endothelin appears to cause a direct release of irANF from rat right atria in vitro. As found for the depressor response in vivo, endothelin is more efficacious in the hypertensive compared with the normotensive atrial preparation. Release of ANF may be important in the hypotensive response to endothelin in vivo.

Functional multiplicity of atrial natriuretic peptide receptors on cultured rat Leydig tumor cells.

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.

Recombinant nematode anticoagulant protein c2, an inhibitor of tissue factor/factor VIIa, attenuates coagulation and the interleukin-10 response in human endotoxemia.

The tissue factor-factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc2. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc2 was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc2 was administered subcutaneously. rNAPc2 elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of prothrombin time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc2 completely blocked the endotoxin-induced thrombin generation, as measured by plasma prothrombin fragment F1+2. The endotoxin-induced effect on fibrinolytic parameters such as plasmin-antiplasmin complexes and plasminogen activator inhibitor type 1 was not affected by rNAPc2. The administration of rNAPc2 attenuated the endotoxin-induced rise in interleukin (IL)-10, without affecting the rise in other cytokines. In conclusion, rNAPc2 is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc2 completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc2 attenuated the endotoxin-induced rise in IL-10, without affecting the rises in other cytokines.

Renin inhibitors containing conformationally restricted P1-P1' dipeptide mimetics.

A series of renin inhibitors containing lactam-bridged P1-P1' dipeptide mimetics based on the ACHPA (4(S)-amino-5-cyclohexyl-3(S)-hydroxypentanoic acid) design was studied. The inhibitors were obtained by aldol addition of various lactams with N alpha-Boc-L-cyclohexylalaninal, followed by Boc group removal and acylation with Boc-Phe-His. The aldol diastereomer having the S configuration at the two newly generated stereogenic centers gave optimal enzyme inhibition. Potency was further enhanced in the gamma-lactam ring series by substitution with small hydrophobic groups to mimic the P1' side chain of the renin substrate. Thus, 2(S)-[(Boc-L-phenylalanyl-L-histidyl)amino]-3-cyclohexyl-1(S)-hydroxyl-1 - (1,5,5-trimethyl-2-oxopyrrolidin-3(S)-yl)propane (34) has an IC50 of 1.3 nM in the human plasma renin assay. A variety of substituents on the lactam nitrogen are tolerated and can be used to vary the physical properties of the inhibitor. By using a model of the human renin active site, the conformation of 34 in the enzyme-inhibitor complex is proposed. This modeled conformation is very similar to the solid-state conformation of 2(S)-[(Boc-L-phenylalanyl-L-histidyl)amino]-3-cyclohexyl-1(S)-hydroxyl- 1-(1-methyl-2-oxopyrrolidin-3(S)-yl)propane (36), the structure of which was determined by single-crystal X-ray diffraction analysis. The most potent ACH-PA-lactam renin inhibitors show good selectivity when assayed against other types of aspartic proteinases. By varying the lactam ring substituents, potent and selective inhibitors of cathepsin D and cathepsin E can be obtained.

Renin inhibitors. Statine-containing tetrapeptides with varied hydrophobic carboxy termini.

A series of statine-containing tetrapeptides, systematically modified at the carboxy terminus with various hydrophobic aromatic groups, is described. These compounds were tested in vitro for their ability to inhibit porcine, human plasma, and purified human kidney renins. These analogues help to define optimal binding aspects in a region of the enzyme that appears to be specific for spatial arrangement of aromatic groups. Replacement of the metabolically labile Phe amide with nonpeptidal groups proved possible while achieving inhibitory potency in the nanomolar range vs. porcine kidney renin. For the compounds 6i, 6m, and 6o, a large discrepancy in potency between the human plasma and the purified human kidney renin assays was observed. This disparity does not appear to be a consequence of a previously proposed plasma binding component.

Atrial natriuretic factor receptors.

All of the actions of atrial natriuretic factor (ANF) can be explained on the basis of ANF's interaction with specific receptors on the plasma membrane of cells in target tissues. These receptors have been identified. Binding to these receptors by ANF is time dependent and specific and occurs with high affinity. Numerous analogues of ANF have been synthesized, and in general, their affinity for ANF receptors corresponds closely to their potency in vitro and in vivo. Considerable progress has been made in physicochemically characterizing ANF receptors by photoaffinity labeling and affinity cross-linking techniques.

Structural and functional characterization of tick anticoagulant peptide (TAP): a potent and selective inhibitor of blood coagulation factor Xa.

The discovery of rTAP has provided a wealth of important scientific information ranging from the structural and kinetic characterization of a novel serine protease inhibitor to the importance of factor Xa in the thrombotic process. The results obtained with this inhibitor thus far have broadened our understanding of hemostasis and thrombosis and ultimately may result in the development of newer and more efficient therapeutic agents to combat one of the leading causes of morbidity and mortality in man today.

Primary prevention of coronary arterial thrombosis with the factor Xa inhibitor rTAP in a canine electrolytic injury model.

The antithrombotic efficacies of the coagulation factor Xa inhibitor recombinant tick anticoagulant peptide (rTAP) and heparin were compared in a canine model of left circumflex (LCX) coronary artery electrolytic lesion. Intravenous infusions of saline (controls), rTAP (50 micrograms/kg/min continuous infusion) or heparin (200 U/kg bolus followed by 2 U/kg/min continuous infusion) were started 60 min prior to the initiation of LCX coronary artery electrolytic injury (150 microA continuous anodal current). All 6/6 saline-treated control animals developed occlusive thrombi at 49.8 +/- 13.6 min after the initiation of vessel injury, and possessed a residual thrombus mass of 20.7 +/- 3.3 mg. In the rTAP treatment group, 4/6 preparations developed occlusive thrombi, but with times to thrombosis delayed significantly compared to both the saline control as well as to the heparin treatment group (202.7 +/- 28.9 min; p < 0.01 to both saline and heparin groups). The remaining 2 rTAP-treated preparations remained patent despite the continued electrical stimulation of the coronary vessel for 5 h. Residual thrombus mass in the rTAP treatment group was reduced markedly compared to the saline control group (4.4 +/- 1.0 mg; p < 0.01). Heparin infusion resulted in a modest but statistically insignificant delay in occlusive LCX coronary artery thrombosis compared to saline controls, with all 6/6 heparin-treated preparations occluding at 79.7 +/- 16.5 min after the initiation of vessel injury. Residual thrombus mass in heparin-treated animals, however, was reduced compared to saline controls (9.4 +/- 1.4 mg; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Thioester chromogenic substrates for human factor VIIa: substituted isocoumarins are inhibitors of factor VIIa and in vitro anticoagulants.

Arginine thiobenzyl esters are convenient chromogenic substrates of factor VIIa (Z-Arg-SBzl, Kcat/KM = 1,600 M-1 s-1) and were used to study the kinetics of inhibition of factor VIIa by several mechanism-based isocoumarin inhibitors of trypsin-like enzymes. Isocoumarin derivatives substituted with a 7-guanidino or 3-isothiureidopropoxy group were good inhibitors of factor VIIa and acted as anticoagulants in human and rabbit plasma. With normal citrated human plasma, 4-chloro-3-ethoxy-7-guanidinoisocoumarin (3) and 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin (ACITIC, 6) prolonged the prothrombin time (PT) ca. two-fold and prolonged the activated partial thromboplastin time (APTT) more than 4.5-fold at 20-30 microM. Both compounds had smaller effects in rabbit plasma. The short half-life of ACITIC and related isocoumarins in plasma should make these compounds uniquely useful as anticoagulants in therapeutic situations where it is desirable to have anticoagulant effects for a short defined time period.

Anticoagulant efficacy and immunogenicity of the selective factor Xa inhibitor antistasin following subcutaneous administration in the rhesus monkey.

The antithrombotic efficacy and duration of action of a single subcutaneous administration of the selective factor Xa inhibitor recombinant antistasin (rATS) was evaluated in a rhesus monkey model of mild disseminated intravascular coagulation. rATS (1 mg/kg) was shown to be fully effective and comparable to standard heparin (1,000 U/kg) in the suppression of thromboplastin-induced fibrinopeptide A generation for at least 5 h following a single subcutaneous administration. The absorption rate of rATS, as measured by ex vivo activated partial thromboplastin times (aPTT), mirrored that of standard heparin exhibiting peak anticoagulant activity between 1 and 2 h post administration. The anticoagulant effects of a single rATS dose lasted for longer than 30 h maintaining an aPTT value at least 2-fold higher than baseline. Repeated subcutaneous administrations of rATS resulted in the generation of fully neutralizing antibodies. These results suggest that specific factor Xa inhibition may be as effective as standard heparin in the treatment of venous thrombosis. Due to its antigenicity however, rATS is probably not suitable for chronic subcutaneous anticoagulant therapy.

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.

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 does not elicit protective immunity in chimpanzees.

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 (HIV-1) was used to immunize chimpanzees. The animals developed high titers of antibodies to p55 as well as to the p24 and p17 mature cleavage products of the core precursor. Virus-neutralizing antibodies were not elicited. The induced immune responses did not prevent establishment of HIV-1 infection following challenge of one immunized chimpanzee with live virus.

Isolation and characterization of the tsetse thrombin inhibitor: a potent antithrombotic peptide from the saliva of Glossina morsitans morsitans.

A potent and specific inhibitor of the human coagulation protease thrombin was identified in salivary gland extracts of the tsetse fly, Glossina morsitans morsitans, an important vector of African trypanosomiasis. This low molecular weight peptide (MW = 3,530 Da as determined by laser desorption mass spectrometry) was purified using a combination of size-exclusion chromatography and reverse-phase, high-performance liquid chromatography, respectively. Amino terminal sequencing of the purified protein reveals no homology to any previously identified serine protease inhibitor or naturally occurring anticoagulant. The tsetse thrombin inhibitor (TTI) is a stoichiometric inhibitor of thrombin, with an apparent equilibrium dissociation inhibitory constant (Ki*) [corrected] of 584 x 10(-15)M. In addition, it is also a potent inhibitor of thrombin-induced platelet aggregation. Like other hematophagous arthropods, tsetse flies appear to have evolved a novel protease inhibitor capable of antagonizing host hemostasis and facilitating blood feeding.