Multicenter, randomized study of the efficacy and safety of intravenous iclaprim in complicated skin and skin structure infections.

Iclaprim is a novel antibacterial agent that is currently in development for the treatment of complicated skin and skin structure infections (cSSSI). Iclaprim specifically and selectively inhibits bacterial dihydrofolate reductase, a critical enzyme in the bacterial folate pathway, and exhibits an extended spectrum of activity against various resistant pathogens, including methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). The objective of this randomized, double-blind phase II study was to compare the efficacy and safety of iclaprim to those of vancomycin in patients with cSSSI. Patients were randomized to receive 0.8 mg iclaprim/kg of body weight, 1.6 mg/kg iclaprim, or 1 g vancomycin twice a day for 10 days. Clinical cure rates for the 0.8- and 1.6-mg/kg-iclaprim treatment groups were comparable to that for the vancomycin treatment group (26/28 patients [92.9%], 28/31 patients [90.3%], and 26/28 patients [92.9%], respectively). Iclaprim also showed high microbiological eradication rates. Iclaprim exhibited an eradication rate of 80% and 72% versus 59% observed with vancomycin for S. aureus, the pathogen most frequently isolated at baseline. Five MRSA cases were observed, four in the 0.8-mg/kg-iclaprim arm and one in the vancomycin arm, and all were both clinically and microbiologically cured. Iclaprim exhibited a safety profile similar to that of vancomycin, an established drug for the treatment of cSSSI. Results from this study indicate that iclaprim is a promising new therapy for the treatment of cSSSI, in particular those caused by S. aureus, including MRSA.

Surface behaviour of bile salts and tetrahydrolipstatin at air/water and oil/water interfaces.

The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.

Inhibition of gastrointestinal lipolysis by Orlistat during digestion of test meals in healthy volunteers.

The inhibition of digestive lipases by the antiobesity drug Orlistat along with lipolysis levels and fecal fat excretion were measured in healthy humans. Orlistat was found to be a powerful gastric lipase inhibitor, achieving 46.6--91.4% enzyme inhibition and thus greatly reducing gastric lipolysis of solid and liquid meals (11--33% of respective controls). Gastric lipase inhibition by Orlistat was extremely fast (half-inhibition time < 1 min). Duodenal lipolysis was reduced significantly by Orlistat given with the solid meal (32.6--37.6% of controls) but was only slightly reduced by Orlistat given with the liquid meal (74.5--100% of controls). Human pancreatic lipase (HPL) inhibition was found to be high (51.2--82.6%), however, regardless of the meal. These paradoxical results were explained when in vitro lipolysis experiments were performed. The rates of HPL inhibition by Orlistat were found to be similar with both types of meals (half-inhibition time 5--6 min), but the preemulsified triglycerides of the liquid meal were rapidly hydrolyzed by HPL before the enzyme was significantly inhibited by Orlistat. With the solid meal, the rate of hydrolysis of the meal triglycerides by HPL was slower than the rate of HPL inhibition by Orlistat. As predicted from the previous results, the effects of Orlistat on fat excretion levels were found to be much greater with the solid (40.5--57.4% of ingested fat) than with the liquid (4.2--18.8%) test meal.

Hydrolysis of dietary fat by pancreatic lipase stimulates cholecystokinin release.

BACKGROUND & AIMS: The hypothesis that cholecystokinin release requires adequate dietary fat digestion in the small intestine was investigated in 10 healthy volunteers, and the consequences of reduced fat hydrolysis on pancreaticobiliary secretions were assessed. METHODS: Fat hydrolysis was inhibited by intraduodenal perfusion of tetrahydrolipstatin, an irreversible lipase inhibitor. An oil emulsion containing 0, 30, 60, or 120 mg tetrahydrolipstatin was perfused. After a 40-minute basal period, a test meal was eaten to stimulate cholecystokinin release and pancreaticobiliary responses. RESULTS: In the control without tetrahydrolipstatin, lipase output increased threefold with meal ingestion and remained doubled for 4 hours. At the ligament of Treitz, free fatty acid concentration averaged 60% of total fatty acids. Increasing doses of tetrahydrolipstatin induced a dose-dependent inhibition of duodenal lipase activity (P < 0.01); 120 mg tetrahydrolipstatin eliminated the postprandial lipase peak activity, free fatty acid levels decreased to < 5% of total fatty acids, and plasma cholecystokinin levels were suppressed by 77% (P < 0.01). Amylase and trypsin outputs were reduced by 77% and 59%, respectively, and bilirubin secretion was virtually abolished (P < 0.01). CONCLUSIONS: These findings show that tetrahydrolipstatin prevents triglyceride hydrolysis and that plasma cholecystokinin release, gallbladder emptying, and pancreatic enzyme secretion require adequate triglyceride digestion. These data also support the concept of negative feedback regulation of cholecystokinin secretion.

Orally active fibrinogen receptor antagonists. 2. Amidoximes as prodrugs of amidines.

The potent and selective GP IIb-IIIa antagonist lamifiban (1, Ro 44-9883) is currently in clinical development as an injectable antithrombotic agent for treating and preventing acute coronary syndromes. However, for secondary prevention of thrombotic occlusions, orally active inhibitors are needed. By means of a prodrug strategy, the modest oral absorption of 1 in mice was improved by a factor of 9. In addition, these studies demonstrated that an amidoxime group can serve as a prodrug functionality for an amidino group. Application of this principle to the structurally related amidino carboxylate 13 led to the amidoxime ester 18 which was absorbed approximately 20 times better, after oral administration to mice, than 13. Due to the modification of the amidino group as well as of the carboxylate group, 18 completely lost its ability to interact with purified platelet GP IIb-IIIa. After oral administration of 18 to rats, dogs, and rhesus monkeys, the bioavailability of the active derivative 13 was 26 +/- 5, 25 +/- 6, and 33 +/- 6%, respectively, and the elimination half-life was 4.1 +/- 1.7, 11.4 +/- 1.1, and 5.1 +/- 1.4 h, respectively. On the basis of these properties, the orally active 18 (Ro 48-3657), a double prodrug of the potent and selective non-peptide GP IIb-IIIa antagonist 13 (Ro 44-3888), was selected as clinical candidate for evaluation as a prophylactic agent in patients at high risk for arterial thrombosis.

Design and synthesis of potent and highly selective thrombin inhibitors.

Thrombin, a serine protease, plays a central role in the initiation and propagation of thrombotic events. An extensive search for new thrombin inhibitors was performed, using an unconventional approach. Screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of (aminoiminomethyl)piperidine (amidinopiperidine) as a weak, but intrinsically selective, thrombin inhibitor. Elaboration of this molecule provided compounds which inhibit thrombin with Ki's in the range of 20-50 nM and with selectivities of 1000-4000 against trypsin. These inhibitor compounds show a new and unexpected binding mode to thrombin. Modification of the central building block and then of one of the hydrophobic substituents led to the discovery of a new family of thrombin inhibitors which has reverted to the former binding mode to thrombin. This last class of compounds shows inhibitory activities in the picomolar range, low toxicity, and a short plasma half life which favors its use for an intravenous application. From this series of thrombin inhibitors, 19f(Ro 46-6240) was selected for clinical development as an antithrombotic agent for intravenous administration.

Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.

TNF-alpha induces changes in endothelial cell functions, such as upregulation of tissue factor, resulting in endothelial procoagulant activity which may play a role in disseminated intravascular coagulation. The procoagulant activity of TNF-alpha-stimulated endothelial cell monolayers was studied in a human ex vivo native (nonanticoagulated) blood flow system using the three thrombin inhibitors recombinant hirudin, Ro 46-6240, and heparin. Under venous blood flow conditions (shear rate 65 s-1) recombinant hirudin, Ro 46-6240, and heparin inhibited fibrin deposition on the endothelial cells by 50% at concentrations of 14, 28, and 412 ng/ml, respectively. The highest tested concentrations of the thrombin inhibitors reduced the postchamber fibrinopeptide A levels from 713 +/- 69 to < 70 ng/ml. Surprisingly, even at relatively high inhibitor concentrations, some local fibrin deposits were found on TNF-alpha-stimulated cells, suggesting that some endothelial cells possess higher procoagulant activity than others. Therefore, the surface expression pattern of tissue factor, the primary initiator of coagulation in this system, was examined by immunogold-silver staining. The results showed that the tissue factor density on the cell surface varied strongly among TNF-alpha-stimulated endothelial cells. Using TNF receptor-selective agonistic mutants of TNF-alpha, it was demonstrated further that the heterogenous surface expression of tissue factor was mediated entirely by the 55-kD TNF receptor and did not involve the 75-kD TNF receptor. We conclude that in this system TNF-alpha induces heterogenous tissue factor expression which may lead to a high local thrombin concentration, such that even in the presence of thrombin inhibitors focal fibrin deposition occurs.

The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit.

Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469). Sf9 insect cells were infected with the corresponding baculovirus, and the produced soluble recombinant proteins were purified using an RGD-like affinity column. The bound receptor fragments were specifically eluted with RGDS and existed as a heterodimeric complex (rec alpha IIbH-t beta 3) with an apparent M(r) of 160,000. In an immunocapture assay, the monoclonal antibody pl-55, which only recognizes the functionally active form of alpha IIb-beta 3, bound to the purified complex. Rec alpha IIbH-t beta 3 specifically bound 125I-fibrinogen with an affinity comparable with that of purified platelet alpha IIb-beta 3. Electron micrographs of rotary-shadowed rec alpha IIbH-t beta 3 showed that the complex had the characteristic globular head, but the two rodlike tails were 4-6 nm shorter than those found in intact alpha IIb-beta 3. Thus, the cysteine-rich repeats of beta 3 are not required for assembly, stability, and functional activity of this integrin.

Tetrafibricin: a nonpeptidic fibrinogen receptor inhibitor from Streptomyces neyagawaensis (I). Its GPIIb/IIIa blockage on solid phase binding assay.

Tetrafibricin is a novel nonpeptidic fibrinogen receptor inhibitor isolated from Streptomyces neyagawaensis NR0577. Its competitive and selective fibrinogen receptor blockage was demonstrated in this study. Tetrafibricin competitively inhibited (Ki = 9.9 nM) the binding of biotinylated fibrinogen to purified active glycoprotein (GP) IIb/IIIa immobilized on plastic plate. When RGDS and tetrafibricin were added in combination, the inhibition was additive. The binding of other RGD-containing proteins, fibronectin and von Willebrand factor, to active GPIIb/IIIa were also completely inhibited by tetrafibricin. The fact that tetrafibricin did not inhibit the binding of von Willebrand factor to GPIb/IX indicates the specific blockage of tetrafibricin for GPIIb/IIIa. Fibrinogen receptor inhibition of tetrafibricin was also confirmed by its ability to inhibit 125I-fibrinogen binding to platelets stimulated with ADP. Because of its competitiveness and specificity, tetrafibricin can be used in a new structural model for the design of fibrinogen receptor inhibitors.

Ro 44-9883, a new nonpeptide glycoprotein IIb/IIIa antagonist, prevents platelet loss during experimental cardiopulmonary bypass.

Extensive contact between blood and artificial surfaces causes platelet activation and depletion. The aim of the study was to test the efficacy of Ro 44-9883, a new nonpeptide, reversible, glycoprotein IIb/IIIa antagonist in preventing platelet count drop in dogs undergoing cardiopulmonary bypass during 2 hours. Twenty-two heparinized dogs were divided into three groups, one control group (n = 9) not treated, one group treated with a low dose of Ro 44-9883 (145 micrograms/kg intravenously, n = 6), and one group treated with a high dose of Ro 44-9883 (870 micrograms/kg intravenously, n = 7). In the control group, platelet counts declined to 53% +/- 15% of initial levels at the start of cardiopulmonary bypass and remained lower than 80% of initial levels during follow-up. Platelet count drop was completely prevented in the Ro 44-9883 high-dose group, whereas it was only partially prevented in the low-dose group. Blood loss was similar in the three groups despite the fact that bleeding times were longer in the Ro 44-9883 high-dose group than in the control group. We conclude that Ro 44-9883, together with heparin as a standard anticoagulant, is highly effective in preventing platelet count drop during cardiopulmonary bypass without causing excessive bleeding.

Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane.

The platelet glycoprotein Ib-IX complex (GP Ib-IX) is essential for the initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib alpha (residues 1-318), containing the ligand binding sites for von Willebrand factor (vWF) and thrombin, as well as the entire human GP Ib alpha were expressed in Chinese hamster ovary cells. The transfected cells secreted a 48- and a 110-kDa protein, respectively, into the supernatant. Both recombinant proteins were purified by immunoaffinity chromatography. The purified proteins bound soluble vWF in the presence of botrocetin as demonstrated in solid-phase binding assays. The dissociation constant (Kd) for 125I-vWF binding to the recombinant 110-kDa protein was 1.2 +/- 0.2 nM as compared with 1.0 +/- 0.3 nM for vWF binding to purified platelet GP Ib-IX. Both recombinant proteins were also retained on thrombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosaccharide chains. A 125-kDa protein was identified in the lysate of cells transfected with the coding sequence for the entire GP Ib alpha. Trypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational removal of the C-terminal transmembrane domain of recombinant GP Ib alpha might have facilitated the secretion of the soluble glycocalicin-like 110-kDa fragment. In addition, flow cytometry and immunofluorescence microscopy demonstrated that the expression of GP Ib alpha alone is sufficient for its incorporation into the cell surface membrane. These data indicate that two soluble fragments of human GP Ib alpha with binding activity for vWF and thrombin can be expressed in mammalian cells and that the incorporation of GP Ib alpha into the surface membrane does not depend on co-expression with GP Ib beta and/or GP IX.

Relationship between tissue factor expression and deposition of fibrin, platelets, and leukocytes on cultured endothelial cells under venous blood flow conditions.

Endothelial cell-mediated coagulation and leukocyte adhesion are processes that might be connected by the generation of thrombin. To examine the interaction of procoagulant and proadhesive activity, cultures of endothelial cells were stimulated with tumor necrosis factor-alpha, which resulted in the surface expression of tissue factor. Subsequent exposure to human nonanticoagulated blood at a shear rate of 100 s-1 in a parallel plate perfusion device led to the deposition of polymerized fibrin, which covered 63% of the endothelial surface. In addition, numerous platelet aggregates (71 per 10 mm cross-section) and leukocytes (53 +/- 6/mm2) were deposited on stimulated endothelial cells, whereas no fibrin and only a few platelet aggregates (4 +/- 1 per 10 mm cross-section) and leukocytes (6 +/- 1/mm2) were detected on control cells. A significant portion of the adherent leukocytes bound to fibrin and platelets. However, when the deposition of fibrin and platelet aggregates was inhibited with the anti-tissue factor antibody HTFI-7B8 by 100% and 86%, respectively, leukocyte adherence remained unchanged (68 +/- 6/mm2). This indicated that leukocytes could efficiently adhere to endothelial cells through direct cell-cell contact independent of both thrombin and deposited fibrin. Moreover, this direct adhesion of leukocytes to the endothelial surface was reduced twofold to threefold when fibrin deposition occurred. These data suggest a relationship between endothelial procoagulant and proadhesive properties in that tissue factor-initiated coagulation may contribute to leukocyte adhesion through the formation of an adhesive fibrin/platelet meshwork but concurrently prevents the adhesive endothelial surface to bind leukocytes at its full capacity.

Effects of heparin, aspirin and a synthetic platelet glycoprotein IIb-IIIa receptor antagonist (Ro 43-5054) on coronary artery reperfusion and reocclusion after thrombolysis with tissue-type plasminogen activator in the dog.

Adjuncts to thrombolytic agents have improved coronary patency and prevented early reocclusion after thrombolysis in acute myocardial infarction. The aim of this study was to compare in a canine thrombolysis model the effects of Ro 43-5054 = N-(N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl)-3- phenyl-L-alanine-trifluor-acetate, a new glycoprotein IIb-IIIa receptor antagonist with aspirin or heparin. Six groups of 10 dogs each were studied. A platelet-rich coronary thrombus was induced in open-chest dogs by electrical stimulation. In addition to recombinant tissue-type plasminogen activator (30 micrograms/kg.min during 60 min), the dogs received 1) saline, 2) heparin 200 U/kg + 50 U/kg.hr i.v., 3) aspirin 10 mg/kg i.v., 4) heparin+aspirin, 5) Ro 43-5054 (3 micrograms/kg.min) and 6) heparin+Ro 43-5054. The overall reperfusion rate was 70% (range, 60-90%) and comparable in all the six groups. During the 120-min observation period, episodes of reocclusion were observed in the absence of antiplatelet therapy (group 1 and 2) irrespective of heparin treatment. Aspirin prevented coronary reocclusion in half of the reperfused dogs (group 3 and 4). However, after reinforcement of the thrombogenic stimulus, 80% of the reperfused dogs treated with aspirin showed reocclusion, whereas none of them reoccluded when treated with Ro 43-5054. Thus, inhibition of platelet activation by the selective, nonpeptidic glycoprotein IIb-IIIa receptor antagonist Ro 43-5054, although without effect on the time to reperfusion, better protected than aspirin against early reocclusion after thrombolytic therapy.

Low molecular weight, non-peptide fibrinogen receptor antagonists.

The tetrapeptide H-Arg-Gly-Asp-Ser-OH (1) (RGDS), representing a recognition sequence of fibrinogen for its platelet receptor GP IIb-IIIa (integrin alpha IIb beta 3), served as lead compound for the development of highly potent and selective fibrinogen receptor antagonists. Replacement of the N-terminal arginine by p-amidinophenylalanine or the Gly moiety by m-aminobenzoic acid led to compounds which are superior to the lead peptide with regard to activity and selectivity for GP IIb-IIIa vs the closely related vitronectin receptor alpha v beta 3. By random screening [(p-amidinobenzenesulfonamido)ethyl]-p-phenoxyacetic acid derivatives have been identified as fibrinogen receptor antagonists. Further structure-activity relationship studies culminated in the preparation of N-[N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl]-3-phenyl-L- alanine (29h, Ro 43-5054) and [[1-[N-(p-amidinobenzoyl)-L-tyrosyl]-4-piperidinyl]oxy]acetic acid (37f, Ro 44-9883), which exhibit very high activity as platelet aggregation inhibitors (IC50s 0.06 and 0.03 microM, respectively, human PRP/ADP) as well as marked selectivity for GP IIb-IIIa vs alpha v beta 3. Since the activity of 37f in dogs declines according to a two-compartment model with an initial phase having a t1/2 of 8 min and a second phase with a t1/2 of 110 min, this compound is a suitable candidate for the development as iv platelet inhibitor.

Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor.

Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 43-5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 43-5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 43-5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 43-5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 43-5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 43-5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 43-5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.

Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa.

This study characterized conformational states of platelet glycoprotein IIb-IIIa (GPIIb-IIIa) and regions of the molecule required for fibrinogen binding. Platelet lysates were passed sequentially over concanavalin A and aminoethylglycine (Aeg)RGDS affinity columns. Approximately 10% of the total GPIIb-IIIa bound to the Aeg-RGDS column. The non-binding GPIIb-IIIa was further purified by S300 gel filtration. Only GPIIb-IIIa which recognized immobilized RGDS bound fibrinogen. The functional difference between the Aeg-RGDS binding GPIIb-IIIa (active) and the S300-purified complex (inactive) suggested that the two populations existed in different conformations. This was confirmed immunochemically and in an assay utilizing endoproteinase Arg-C. Active GPIIb-IIIa was heavily degraded by Arg-C, whereas inactive GPIIb-IIIa was highly resistant to degradation. Receptor occupancy by RGDV or peptidomimetic inhibitors prevented degradation of regions of the active complex and stimulated hydrolysis of the inactive receptor such that the two populations yielded fragments of identical electrophoretic mobility. Induction of hydrolysis of inactive GPIIb-IIIa required 15-fold higher concentrations of RGDV than protection of the active complex. Upon removal of inhibitor, fragments generated from either active or inactive GPIIb-IIIa bound fibrinogen. The ability of carboxypeptidase Y to digest inhibitor-protected GPIIb-IIIa was also examined. GPIIb was cleaved to a 58-kDa NH2-terminal fragment, whereas GPIIIa remained essentially intact. The complexed fragments bound fibrinogen with similar affinity as intact GPIIb-IIIa. This binding was inhibited by both RGDV and HHLGGAKQAGDV peptides. These data suggest that: 1) purified active and inactive GPIIb-IIIa exist in different conformations and have different affinities for RGDV; 2) certain peptidomimetic inhibitors (Ro 42-1499 and Ro 43-5054) alter the conformation of inactive GPIIb-IIIa; 3) GPIIIa and a 58-kDa NH2-terminal fragment of GPIIb alpha form a high affinity fibrinogen binding complex.

Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin.

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C] tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the delta-lactone Ro 40-4441 in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152 ... His-263 ... Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.