Search

Pullulan microspheres containing cyclodextrin (CyD) were obtained by chemical crosslinking with epichlorohydrin of an alkaline solution of pullulan (Pul) and alpha-, beta- or gamma-CyD. The amount of alpha-, beta- and gamma-CyD in microspheres was 120, 156, and 138 micromol/g, respectively, as determined from the percentage of iodine incorporated in the hydrophobic cavity of CyD's. Microspheres were packed in a glass column and the liquid chromatographic behaviour by isocratic elution of different drugs or typical organic compounds (TOC), taken as model drugs, was investigated. The increase of the retention volume (V(R)) of each compound, depending on the interaction(s) between CyD's cavity and the considered molecule, is characterized by a broadening of the peaks. The interaction coefficient K, corresponding to the ratio between the V(R) value of each tested molecule on Pul-alpha-, Pul-beta- and Pul-gamma-CyD active stationary phase and the V(R) value of benzoic acid on St/maltodextrin neutral stationary phase, was determined. According to K values, the accurate prediction can be done on the potential drugs to be conditioned in suitable CyD cavity. Values of K allow to anticipate the release profiles of drugs considered.

The pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor, is one of the most extensively studied globular proteins. It has proved to be a particularly attractive and powerful tool for studying protein conformation as well as molecular bases of protein/protein interaction(s) and (macro)molecular recognition. BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase-like serine (pro)enzymes endowed with very different primary specificity. BPTI reacts rapidly with serine proteases to form stable complexes, but the enzyme: inhibitor complex formation may involve several intermediates corresponding to discrete reaction steps. Moreover, BPTI inhibits the nitric oxide synthase type-I and -II action and impairs K+ transport by Ca2+-activated K+ channels. Clinically, the use of BPTI in selected surgical interventions, such as cardiopulmonary surgery and orthotopic liver transplantation, is advised, as it significantly reduces hemorrhagic complications and thus blood-transfusion requirements. Here, the structural, inhibition, and bio-medical aspects of BPTI are reported.

A study of the diffusion of parabens from topical formulations is presented here. In particular, four different topical formulations, namely, a water-in-oil emulsion, an oil-in-water emulsion, and two hydrophilic gels (Pemulen gel and Carbopol gel) were produced, containing a mixture of three common parabens, namely, methylparaben (MP), ethylparaben (EP), and propylparaben (PP). An analytical method based on liquid extraction, followed by reversed-phase HPLC for the quantitative determination of MP, EP, and PP, was developed. The method allowed good separation of paraben mixtures and high percentages of recovery (> than 97%). The diffusion kinetics of parabens from the produced formulations was determined by an in vitro system based on a Franz cell assembled with a synthetic membrane, followed by a reversed-phase HPLC analytical method. The comparative study demonstrated that, in the case of emulsions, diffusion coefficients are a function of the substituent of preservatives: the higher the solubility, the higher the diffusion of parabens. On the contrary, in the case of the hydrophilic gels, the higher the parabens solubility, the lower the diffusion coefficients. The method described here could represent a means of controlling the extent of diffusion of parabens from topical formulations in order to minimize percutaneous absorption and to control the availability of microbes.

Background: Among the possible variables associated with cytokine activation in hemodialysis, filter membrane has been reported to be a major factor and monocytes adherent to the membrane have been suggested as a possible source for cytokine synthesis., Methods: In order to exclude variables other than the direct cell-to-membrane interaction, suspensions of peripheral blood monocytes isolated from donors' blood were incubated for 180 min at 37 degrees C in Petri dishes coated with cuprophan (Cu) or polyacrylonitrile (AN69S) or polysulfone (PS). Total RNA was purified, reverse transcribed in cDNA and amplified by polymerase chain reaction (PCR) primed with specific oligomers for determining IL-1beta and TNF-alpha gene expression. For Western blot analysis, cell homogenates and supernatants were electrophoresed and transferred to a polyvinylidene difluoride membrane and the membrane was then incubated with polyclonal antibodies specific for the detection of IL-1beta and TNF-alpha., Results: Semi-quantitation of targets for PCR (using the technique of limiting dilutions and referring to actin and glyceraldehyde-3-phosphate dehydrogenase as noninducible molecule) revealed a comparable increase at 180 min (p < 0.05) in IL-1beta and TNF-alpha mRNA in monocytes incubated with polyacrylonitrile, PS and Cu membranes. In 2 of 10 experiments pro-IL-1beta was detected in monocytes interacting with Cu, PS and AN69S membrane. However, the extent of extracellular release of mature protein was greater for Cu., Conclusion: Some dissociation between transcriptional and translational events was detected in experiments using donors' blood in vitro. More specifically, synthetic membranes (both polyacrylonitrile and PS) were found to be as active as Cu in inducing IL-1beta and TNF-alpha mRNA expression, but less effective in promoting the extracellular release of proinflammatory cytokine products., (Copyright 2002 S. Karger AG, Basel)

The aim of the present paper was to study production of methacrylate microparticles for the delivery (administration) of ascorbic acid via the oral route. Vitamin C is an important antioxidant that may be involved in the reduction of the risk of certain types of cancer, such as colorectal cancer. As polymers different acrylic compounds were considered, namely Eudragit RL, L and RS. Spray-drying was used as preparation method of vitamin C/Eudragit microspheres. Microspheres were first characterized by size and morphology by scanning electron microscopy, then in vitro release kinetics by mean of dialysis method were studied. Although the produced microparticles were unable to slow down the release of the drug with respect to the free form of ascorbic acid, these microspheres showed a good morphology and size distribution that permit to propose them as candidate for the delivery of vitamin C as associated therapy in the treatment of colorectal cancer by oral route.

Uteroglobin (UG) is a multifunctional protein with anti-inflammatory/immunomodulatory properties. The UG gene is located on the long arm of chromosome 11 (11q12.3-q13.1) in a region linked to some immune disorders. A guanine-adenine substitution at position 38 (A38G) has been found in the noncoding region of exon 1 that is significantly correlated with an increased risk of developing immune-mediated diseases. Recently an experimental model of UG knockout mice showed that in mice, UG deficiency causes severe glomerulopathy with mesangial deposition of IgA-fibronectin complexes. To detect the presence of polymorphisms in the UG coding sequence, the DNA of 109 patients with IgA nephropathy (IgAN), and 32 patients with systemic lupus erythematosus (SLE) were tested for the nucleotide sequence of all three UG exons by heteroduplex analysis. We detected heterozygous DNA only for exon 1 due to the A38G substitution, as confirmed by sequencing. We tested for A38G polymorphism, by restriction endonuclease digestion (Sau96I), both in SLE patients and in IgAN patients. Twenty patients with either membranous nephropathy (12) or focal and segmental glomerular sclerosis and 120 healthy subjects served as controls. Compared with both healthy controls and non-IgA control patients, the frequency of the 38A allele was significantly higher in SLE patients (38 of 64 alleles versus 89 of 240 alleles, p = 0.002, and versus 7 of 40 alleles, p < 0.001). IgAN patients showed an allelic distribution similar to both control groups. A subgroup of 18 IgAN patients undergoing renal replacement therapy because of end-stage renal disease showed a significant increase in 38A allele frequency (5 of 36 38G alleles versus 31 of 36 38A alleles, p < 0.001). UG is an immunomodulatory agent that is able to (a) inhibit the activity of several phospholipase A2 (PLA2s), (b) interfere with the function of both neutrophils and monocytes, and (c) prevent immune recognition, perhaps by masking surface antigens. This could account for the role this molecule plays in SLE. The A38G polymorphism is located within a region corresponding to the rat minimal promoter that proved to be important in the transcriptional regulation of UG. Although the significance of any alterations in the UG exon 1 noncoding region in humans has yet to be clarified, initial evidence suggests that it may alter the control of immune response and of inflammation.

Purpose: A series of 5'-esters of N6-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A1 agonists. Log P values, stability, affinity, and activity toward human adenosine A1 receptors were evaluated., Methods: An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5'-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A1 receptor expressed by N6-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3'-5'-cyclic adenosine monophosphate, performing competitive binding assays., Results: All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5'-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A1 receptor of the intact prodrugs., Conclusions: We propose 5'-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible candidates for CPA prodrugs.

Gabexate mesylate is a non-antigenic synthetic inhibitor of trypsin-like serine proteinases that is therapeutically used in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. Considering the structural similarity between gabexate mesylate and arginine-based inhibitors of trypsin-like serine proteinases, the effect of gabexate mesylate on human and bovine mast cell tryptase action was investigated. Values of the inhibition constant (K(i)) for gabexate mesylate binding to human and bovine tryptase were 3.4 x 10(-9) M and 1.8 x 10(-7) M (at pH 7.4 and 37.0 degrees ), respectively. Furthermore, gabexate mesylate inhibited the fibrinogenolytic activity of human tryptase. On the basis of the available x-ray crystal structure of human tryptase, the possible binding mode of gabexate mesylate to human and bovine tryptase was analyzed. Human tryptase inhibition by gabexate mesylate may account for the reported prevention of inflammation, erosion, and ulceration of skin and mucosae.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.

A comparison of the effect of isothiocyanates and nitriles derived from some glucosinolates, namely, epi-progoitrin, sinalbin, glucotropaeolin, glucocheirolin, and glucoraphenin, on human erythroleukemic in vitro cultured cells was studied. Many studies have in fact evidenced that a consumption of vegetable containing glucosinolates could reduce the development of colorectal cancer. In the experimental conditions used, the production of isothiocyanates and nitriles from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The obtained results demonstrated that in general nitriles are considerably less potent than the corresponding isothiocyanates in inhibiting cancer cell growth. Particularly, the isothiocyanates inhibitory activity on K562 cells growth is higher in the case of products derived from epi-progoitrin, glucotropaeolin, glucoraphenin, and glucocheirolin; while for nitriles the higher activity in inhibiting K562 cells growth is showed by sinalbin-derived product. Considering the antiproliferative activity found for isothiocyanates and nitriles, further studies will be aimed to the possible application of glucosinolate-derived products as chemopreventive cancer agents for the reduction of colorectal cancer.

The goal of the present study was to evaluate the influence of the formulation and operating conditions on pellet preparation by pan technique. To this end, a new pelletization process, typified by the application of powdered drug on sugar-based cores using the GS coating system was studied. Inert cores were intermittently treated with micronized drug powder and adhesive solution. This treatment led to the formation of multiple layers of drug particles around an inert core resulting in the production of pellets that can further be coated by different polymers to obtain modified release formulations. Different procedures have been used to evaluate a series of important parameters such as initial core weight; speed of powder application; speed, type, and position of the atomizers; atomization degree; temperature; and air cap. Good yield of drug layering was obtained by adjusting the quantity of both the drug powder to apply and the binder solution. Pellets obtained following the optimal operating conditions (defined in a pre-formulation study) were film coated with the acrylic polymer Eudragit L30D in order to produce a model formulation consisting of enteric polymer-coated pellets containing ibuprofen. During its preparation, the formulation showed no degradation of the drug; moreover, a low percentage of residual humidity was obtained, indicating that this system is very efficient for the production of highly stable formulations. This study showed the good performance of the GS automated pan-coating system in obtaining enteric coated pellets prepared by powder layering technique using aqueous solutions.

The production and characterization of cationic microparticles based on Eudragit RS and cationic agents (i.e. a cationic acrylic polymer and three different cationic surfactants) for the delivery of nucleic acids is here described. It was found that morphological and dimensional characteristics of microparticles were influenced by the type and concentration of cationic agent employed and by some experimental parameters such as stirring speed, emulsifying agent and type of rotor. The desoxiribonucleotide Defibrotide (DFT) was associated with positively charged microparticles and its in vitro release kinetics from microparticles were determined. A study of the in vitro toxicity of cationic microparticles on cultured human cell line K562 was also performed, demonstrating that DDAB(18) microparticles display very low cytotoxicity.

Leukotrienes (LT) of the 5-lipoxygenase pathway constitute a class of potent biological lipid mediators of inflammation implicated in the pathogenesis of different models of experimental glomerulonephritis. The key enzyme, 5-lipoxygenase (5-LO), catalyses oxygenation of arachidonic acid to generate the primary leukotriene LTA4. This LT, in turn, serves as a substrate for either LTA4 hydrolase, to form the potent chemoattractant LTB4, or LTC4 synthase, to produce the powerful vasoconstrictor LTC4. To investigate the potential role of LT in the pathogenesis of human glomerulonephritis with nephrotic syndrome, we examined the gene expression of 5-LO and LTA4 hydrolase in renal tissue of 21 adult patients with nephrotic syndrome and 11 controls. The patients consisted of 11 cases of membranous nephropathy (MN), seven focal and segmental glomerulosclerosis (FSGS), two non-IgA mesangial glomerulonephritis and one minimal change disease. Total RNA purified from renal tissue was reverse transcribed into cDNA and amplified with specific primers in a polymerase chain reaction (RT-PCR). Eight patients' renal tissue, four MN and four FSGS, co-expressed 5-LO and LTA4 hydrolase. In situ hybridization analysis revealed 5-LO expression and distribution limited to the interstitial cells surrounding the peritubular capillaries. Comparative clinical and immunohistological data showed that these eight patients had impaired renal function and interstitial changes that significantly correlated with 5-LO expression. These findings suggest that leukotrienes may play an important role in the pathogenesis of MN and FSGS. These results are also relevant to elucidating the pathophysiologic mechanisms which underlie progression to renal failure in these diseases.

A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.

This paper describes the use of oxidized dextran as a cross-linker for the preparation of gelatin microspheres. Microspheres were obtained by a thermal gelation method and their dissolution kinetic was examined. In order to find evidence of sugar mediated cross-linking, swelling tests and gelatin microspheres dissolution experiments were performed. The obtained results indicated that oxidized dextran can form a cross-linked gelatin network which can reduce the dissolution of gelatin. More interestingly, gelatin microspheres treated by both native and oxidized dextran slow down, even if to a different extent, the release of the antitumor drug TAPP-Br used as a model compound. Taken together, our results suggest that oxidized dextran could be an interesting means to cross-link gelatin microspheres allowing the use of this delivery formulation for controlled release of drugs.

The organic solvents employed in liposome preparation, such as chlorinated solvents, diethyl ether or methanol, although usually removed by evaporation, may remain as traces in the final formulation representing a possible risk for human health and influencing the stability of the vesicles. In order to solve the above mentioned disadvantages, this paper describes the use of different organic solvents, namely ethanol, ethyl acetate and two mixtures of ethanol/ethyl acetate, for the production of liposomes by the reverse phase evaporation technique. After preparation, liposomes were extruded through polycarbonate filters and then characterized by size and encapsulation efficacy. As model drugs retinyl acetate and sodium cromoglycate have been used. The association yield for the hydrophilic drug and the encapsulation yield for the hydrophilic model drug were found, in all the liposome preparations, to be satisfactory. In particular, the use of more polar organic solvents (with respect to diethyl ether) appear to enhance the encapsulation of the hydrophilic drug, sodium cromoglycate. The use of alternative organic solvents to diethyl ether can be proposed with the aim of reducing the toxic problems associated with the presence of residual traces of organic solvents in the final liposome formulation.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.

In the present paper the production and characterization of liposomes are described as a specialized drug delivery system for chromomycin. Liposomes were prepared by the reverse phase evaporation technique followed by extrusion through polycarbonate filters; afterwards the vesicles were characterized in terms of dimensions, morphology and encapsulation efficacy. The aim of this work was to produce a drug delivery system able to reduce the toxicity problems related to the administration of this drug. The analysis of the in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that ionic and neutral liposomes containing chromomycin are 1.5 and 7-fold more effective respectively as compared to the free drug. Based on these results and taking into account the increased solubility of the drug in this system, liposomes could represent a promising drug delivery system for use in the experimental therapy using chromomycin.

Considering the structural similarity between gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, and L-arginine, the effect of gabexate mesylate on the nitric oxide (NO) pathway has been investigated. Gabexate mesylate inhibits competitively constitutive and inducible NO synthase (cNOS and iNOS, respectively), with Ki values of 1.0 x 10(-4) M and 5.0 x 10(-3) M, respectively, at pH 7.4 and 37.0 degrees C. However, gabexate mesylate is not an NO precursor. Moreover, like other NOS inhibitors, gabexate mesylate increases iNOS mRNA expression in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Finally, gabexate mesylate inhibits dose-dependently nitrite production (i.e. NO release) in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Thus, this drug should be administered under careful control, since enzyme inhibition may occur also in vivo.

Leukotrienes (LTs), the end products of the eicosanoid pathway released during inflammation, are markers of polymorphonuclear cell and monocyte activation. The present study focused on the possibility that 5-lipoxygenase (5-LO), the key enzyme for LT synthesis, was involved in the interaction between blood and the hemodialysis (HD) membrane. 5-LO gene expression was examined by reverse transcriptase polymerase chain reaction (RT-PCR) in samples of mononuclear cells isolated from peripheral blood withdrawn at the start and at 15 min of HD from 10 chronic HD patients, 5 treated with Cuprophan and 5 with polymethylmethacrylate (PMMA) membrane. An increased 5-LO gene expression was detected at 15 min in 4 of 5 patients using the Cuprophan membrane but in none of the 5 PMMA treated patients. Our results showed for the first time that the interaction between blood and the HD membrane upregulates 5-LO messenger ribonucleic acid (mRNA).

Along with the numerous technological improvements in molecular biology, polymerase chain reaction, which permits analysis of sequences of a very small amount of biological material, enables evaluation of hemodialysis-induced gene transcription of inflammatory cytokines. Blood samples drawn from 22 hemodialysis patients, treated with cellulose-derived or synthetic membranes, were collected at 0 and 15 min of hemodialysis. Total RNA, purified from mononuclear cells, was reverse transcribed and cDNA amplified by polymerase chain reaction primed with specific oligomers in order to determine tumor necrosis factor alpha (TNF alpha), interleukin (IL) 1 beta and IL6 gene expression. Plasma samples were collected at 0 and 180 min for detection of mature cytokines by enzyme immunoassay with plates pre-coated with monoclonal antibodies to TNF alpha, IL1 beta and IL6. A significant increase in TNF alpha mRNA was detected at 15 min of hemodialysis in 12 of 22 patients: 5 of 9 treated with cuprophan; 3 of 3 with cellulose triacetate; 3 of 5 with polysulfone, and only 1 of 5 treated with polymethyl-methacrylate membranes. A parallel increase in IL1 beta or IL6 mRNA was detected, and significant relationships were found between TNF alpha and IL1 beta (p < 0.001), and IL1 beta and IL6 gene expression (p < 0.05). Increased levels of mature TNF alpha and IL1 beta molecules in plasma were detected in the majority of patients showing an increased cytokine gene expression. However, the absolute amount of cytokine mRNA transcription at 15 min did not predict the levels of mature molecules reached in plasma at 180 min. Cytokine mRNA transcription is quite common at the beginning of a dialysis run. Possibly due to intracellular degradation of critical sequences of cytokine mRNA, gene expression does not necessarily imply translation into mature protein. It is suggested that mechanisms related to cell-to-cell interaction, which may possibly involve procytokine biology, are needed to drive phenomena of cytokine activation to clinical effectiveness.

Amiloride, a diuretic and antihypertensive drug, inhibits Na+ transporting systems, diamine oxidases and the human urinary plasminogen activator. Present results indicate that amiloride is a competitive inhibitor of mouse brain nitric oxide synthase (NOS; Ki = 4.5 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not a nitric oxide (NO) precursor, representing a case for enzyme cross-inhibition. The decreased levels of NO, as a consequence of NOS inhibition, might induce some clinically-adverse amiloride reactions, such as an unexpected reduced antihypertensive effect.

Epidemiological and pharmacological studies have shown that colorectal cancer development could be reduced by consuming vegetables that contain glucosinolates. In view of this the effect of some glucosinolates and their isothiocyanate (ITC)-derived products on in vitro cell growth was studied. We report the isolation and characterization of ITCs derived from glucosinolates by using HPLC, GC-MS, and NMR techniques. The in vitro activity of ITCs on human erythroleukemic K562 cells has been investigated by using two alternative approaches: the in situ and pre-mix methods. No differences in antiproliferative activity were found comparing the effect of ITCs produced either of these methods. In the experimental conditions used, the production of ITCs from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The ITCs' inhibitory activity on K562 cells growth is particularly evident in the cases of ITCs derived from sinigrin, progoitrin, epi-progoitrin, glucotropaeolin and glucocheirolin. Finally, the antiproliferative activity of the ITCs obtained from glucoraphenin, taken as an example, was determined on other tumor cell lines with a different origin and hystotype. Considering the antiproliferative activity found for ITCs these compounds could be considered potentially responsible for the reduction of colorectal cancer associated with diets rich in cruciferous vegetables. Further studies will be aimed at the possible application of glucosinolate-derived products as chemopreventive cancer agents.

p-Aminobenzamidine competitively inhibits bovine trypsin, human and bovine thrombin, and human plasmin, all of which act on substrates containing preferentially the L-arginyl side chain at their P1 position. Considering the structural and functional similarity between p-aminobenzamidine and the L-arginyl side chain in trypsin-like serine proteinases, we investigated the interaction of p-aminobenzamidine with mouse brain nitric oxide synthase (NOS), which uses L-arginine as the substrate for generating NO and L-citrulline. p-Aminobenzamidine is a competitive NOS inhibitor (Ki = 1.2 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not an NO precursor. Therefore, p-aminobenzamidine affects the NO production and the trypsin-like serine proteinase action.

This paper describes the production and characterization of biodegradable microparticles containing tetracycline, designed for periodontal disease therapy. The influence of production parameters on microparticle characteristics and antibiotic release modality was studied. Microparticles were made by using different preparation procedures and different polyesters, namely poly(L-lactide), [L-PLA] poly(DL-lactide), [DL-PLA] and poly(DL-lactide-co-glycolide) 50:50, [DL-PLG]. A double emulsion preparation method together with a concentrated salt solution as external phase gave the best results in terms of tetracycline incorporation efficacy. In vitro release experiments demonstrated that tetracycline is slowly and appropriately released from microparticles. Release kinetics were found to be influenced by the type of polymer utilized for microparticle production. In vitro experiments, simulating in vivo conditions were carried out for up to 30 days. Only DL-PLG microparticles showed significant changes in their morphology, whereas L-PLA and DL-PLA were found almost intact after the same period of time.

The objective of this study was to determine intradialytic blood levels of nitric oxide (NO), in patients undergoing chronic haemodialysis. This was done by detection of nitrosylhaemoglobin by a sensitive technique of spin trap electron paramagnetic resonance at 0, 5, 15, 60, 180 and 240 min of a 4-h standard bicarbonate dialysis, using the same dose (6000 U) of heparin and different dialysis membranes. The study group included 12 patients treated with cellulose-derived dialysis membranes (nine with cuprophan and three with cellulose triacetate) and 10 patients treated with synthetic membranes (five with polysulfone and five with polymethylmethacrylate). Control groups included 11 normal subjects and six patients with end-stage renal failure who were receiving intermittent peritoneal dialysis. Basal blood levels of nitrosylhaemoglobin in haemodialysis patients were significantly higher than normals, but similar to peritoneal dialysis patients. A significant increase (P < 0.01) in nitrosylhaemoglobin level was detected at 15 min of haemodialysis irrespective of the membrane used. A decrease to basal levels at 180 min was observed in all but two cuprophan-treated patients who, in contrast to the others, had a symptomatic hypotension at the end of the session and a further increase in blood nitric oxide. Patients undergoing peritoneal dialysis did not show any change in blood levels of nitrosylhaemoglobin during the first 180 min of the procedure. Thus, a constant increase in nitrosylhaemoglobin levels was observed early in haemodialysis, but not in peritoneal dialysis patients. Very preliminary evidence was obtained for a role of nitric oxide in the vascular instability at the end of haemodialysis in a few patients who had hypotensive episodes.

The crystal structure of bovine alpha-chymotrypsin (alpha-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 A resolution (R-factor = 0.18). The proteinase:inhibitor complex forms a compact dimer (two alpha-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between alpha-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P1 residue of BPTI, however, does not occupy the alpha-CHT S1 specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217.

The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

The presence of proteins (albumin and globulins) in lymphedematous tissue not only gives rise to colloidosmotic pressure but also produces an electrostatic charge endowing the proteins with individual features and different migration rates. The working hypothesis of the experimental study is to transfer lymph proteins from the upper fascia accumulation area to a subfascial drainage area by subjecting them to an adequate difference in potential. A double chamber, variable volume system with separation wall able to contain a 1 cm square of muscle fascia, was designed and built; the aim of the apparatus was to reproduce the subcutaneus zone separated by the fascia interposition, from the muscle-vascular zone. At the system was applied a variable electric field in six different experiments: 4 using porous synthetic membranes and 2 using human muscle fascia.

The inhibitory effect of 4',6-diamidino-2-phenylindole (DAPI) and 6-amidinoindole on the catalytic properties of bovine beta-trypsin (trypsin), human alpha-thrombin (thrombin) and porcine pancreatic beta-kallikrein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Next, the X-ray crystal structure of the trypsin:DAPI complex was solved at 1.9 A resolution (R = 0.161). Over the whole pH range explored, values of the association inhibition constant (Ki) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikrein are higher than those found for benzamidine association, suggesting a binding mode of DAPI to the enzyme primary specificity pocket-based on the indole moiety of the inhibitor. On lowering the pH from 5.5 to 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamidine binding to trypsin, thrombin and kallikrein reflects the acidic pK shift of the Asp189 invariant residue, present at the bottom of the primary specificity subsite of the serine proteinases considered, from 4.5, in the free enzyme, to 3.7, in the proteinase:inhibitor complexes. Inspection of the refined crystal structure of the trypsin:DAPI complex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those reported for related serine (pro)enzyme/inhibitor systems.

The use of supercritical fluid extraction (SFE) as an alternative to liquid extraction was examined for the isolation of vitamin A palmitate and vitamin E acetate from cream and lotion preparations. Investigation of the factors controlling the extraction efficiency in SFE indicated that vitamin recoveries were affected mainly by the extraction pressure and by the degree of sample dispersion. The vitamins were analysed by high-performance liquid chromatography after a 30-min extraction of the cosmetic product with supercritical carbon dioxide at 40 degrees C and at a pressure of 250 atm. Compared with conventional liquid extraction SFE produced slightly lower recoveries (> 91.6%) but afforded a more effective purification of the cosmetic matrices. Moreover, SFE minimized sample handling and the use of harmful solvents and provided mild extraction conditions for the analysis of the labile vitamins. Vitamin E acetate and vitamin A palmitate were assayed in commercial cream and lotion formulations using the proposed SFE technique.

This paper describes how the use of different in vitro experimental systems can influence the determination of (a) the drug release profile from microparticles and (b) the interpretation of the release mechanism(s). We employed, as model dosage form, the Parlodel LA, a recently marketed microsphere system especially designed for bromocriptine-controlled delivery. The release kinetics of bromocriptine from microspheres were determined by using two different experimental approaches: a dialysis method and a flow-through cell method. From the comparison of the obtained data it clearly appears that different in vitro experimental models lead to distinct results in terms of drug availability. On the contrary both series of data can be convincingly fitted with the same mathematical equation, giving almost identical results in terms of postulated release mechanism. Taken together these results indicate that different experimental approaches should always be employed to determine drug release kinetics from microparticles in order to obtain more reliable information on the therapeutic dose (bioavailable drug, for in vivo experiments) and on the uniformity of different batches of microspheres.

The preparation and characterization of gelatin microspheres (GMs) containing the antitumour aromatic tetra-amidine TAPP-Br and its release characteristics from microspheres are described. Spherical gelatin microspheres, showing a high percentage of drug encapsulation (over 95 per cent) and an average diameter of 16 microns were obtained by a coacervation method. In addition, the effects of various reaction parameters on the production and chemical hardening of GMs have been investigated. In order to compare the TAPP-Br release profiles from GMs prepared with different procedures, in vitro studies were carried out. The biological effect of TAPP-Br released from gelatin microspheres was determined on 'in vitro' cultured tumour cell lines, showing that, after encapsulation, TAPP-Br still retains antiproliferative activity comparable with that exhibited by the free drug. Taken together, the results obtained suggest that gelatin-based microspheres offer excellent potential as carriers for the controlled release of polyamidines in anticancer chemotherapy.

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin with apparent dissociation constants of 3.0 x 10(-10) M and 4.1 x 10(-7) M, at pH 8.0 and 21 degrees C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M(r) of about 6.7 kDa. The P1-P1' reactive site bond has been tentatively identified at position Arg20-Ile21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.

Binding of the human recombinant secretory leukocyte proteinase inhibitor (SLPI) [native and with the methionyl residues at positions 73, 82, 94 and 96 of domain 2 oxidized to the sulfoxide derivative (Met(O) SLPI)] to bovine alpha-chymotrypsin (alpha-chymotrypsin) [native and with the Met192 residue converted to the sulfoxide derivative (Met(O) alpha-chymotrypsin)] as well as to native bovine beta-trypsin (beta-trypsin), which does not contain methionyl residues, has been investigated between pH 4.0 and 8.0, and between 10.0 degrees C and 30.0 degrees C, from thermodynamic and/or kinetic viewpoints. By increasing the number of oxidized methionyl residues present at the proteinase:inhibitor contact interface (from 0 to 3), the adducts investigated are increasingly destabilized and the relaxation time of the complexes into conformers less stable is enhanced. On the other hand, the selective oxidation of methionyl residues of SLPI and alpha-chymotrypsin, by reaction with chloramine T, does not affect the proteinase inhibition recognition mechanism. Therefore, even though conformational changes may occur in the conversion of native SLPI and native alpha-chymotrypsin to their Met(O) derivatives, a localized steric hindrance can be considered as the main structural determinant accounting for the reported results.

Values of the association equilibrium constant (Ka) for the binding of the native and of the cyanogen bromide-cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone-52]-52,53-seco-BPTI, respectively) to neuraminidase-treated porcine pancreatic beta-kallikrein-B (kallikrein) and bovine alpha-chymotrypsin (chymotrypsin) have been determined between pH 4.0 and 9.0, at 20.0 degrees C. Over the whole pH range explored, native BPTI and [Hse lactone-52]-52,53-seco-BPTI show the same affinity for kallikrein. On the other hand, the affinity of [Hse lactone-52]-52,53-seco-BPTI for chymotrypsin is higher, around neutrality, than that found for native BPTI by about one order of magnitude, converging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0, (i) the decrease in affinity for the binding of native BPTI to kallikrein and chymotrypsin, as well as for the association of [Hse lactone-52]-52,53-seco-BPTI to kallikrein, reflects the acidic pK shift, upon inhibitor association, of a single ionizing group; and (ii) the decrease of Ka values for [Hse lactone-52]-52,53-seco-BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non-equivalent proton-binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long-range structural changes in [Hse lactone-52]-52,53-seco-BPTI are energetically linked to the chymotrypsin:inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.

Catalytic and inhibitor binding properties of bovine alpha-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 alpha-chymotrypsin), have been examined relative to the native enzyme (alpha-chymotrypsin), between pH 4.5 and 8.0 (mu = 0.1), and/or 5.0 degrees C and 40.0 degrees C. Values of kcat, k+2 and/or k+3 for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 alpha-chymotrypsin are similar, as well as values of Km for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, Ks and Km values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 alpha-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).

The effect of pH and temperature on kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor; BPTI) as well as bovine and porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor; bovine and porcine PSTI, respectively) to human cathepsin G (EC 3.4.21.20) has been investigated. The affinity of the macromolecular inhibitors examined for cathepsin G is characterized by an endothermic, entropy-driven, behaviour, and shows the following trend: BPTI > bovine PSTI > porcine PSTI. The affinity difference of BPTI as well as of bovine and porcine PSTI for cathepsin G is mostly accounted for by changes in the values of the apparent dissociation rate constant for the proteinase:inhibitor complex destabilization. On increasing the pH from 4.5 to 9.5 (at 25.0 degrees C), the affinity of BPTI, as well as bovine and porcine PSTI for cathepsin G increases thus reflecting the acidic-pK shift of the His-57 catalytic residue from approximately 6.9 in the free enzyme to approximately 5.0 in the serine proteinase:inhibitor complexes. The BPTI as well as the bovine and porcine PSTI binding properties of cathepsin G have been analyzed in parallel with those of related serine (pro)enzyme/macromolecular inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI as well as that of bovine and porcine PSTI to cathepsin G has been related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).

In the present study are reported the differential DNA binding activity of the anti-tumor polyamidine tetra-p-amidinophenoxyneopentane (TAPP-H) and its 2'-halo derivative (TAPP-Br), and their effects on the binding of the recombinant Epstein-Barr virus (EBV) nuclear antigen to a synthetic oligonucleotide mimicking the target DNA sequence present in the EBV genome. In addition, the proliferation kinetics and cell cycle analysis of human leukemia K562 cells treated with TAPP-H and TAPP-Br are reported. The possible in vivo relationship between DNA binding affinity and cytotoxicity is also discussed.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin >> human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.

A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI-2), has been isolated from white mustard (Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin, with dissociation constants (Kd) of 1.6 x 10(-10) M and 5.0 x 10(-7) M, respectively, at pH 8.0 and 21 degrees C, the stoichiometry of both proteinase-inhibitor complexes being 1:1. The amino acid sequence of MTI-2, which was determined following S-pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors.

The inhibitory effect of mono-, bis-, tris- and tetra-benzamidine structures (benzamidine, DAPP, TAPB and TAPP, respectively) on the catalytic properties of human alpha-, beta- and gamma-thrombin (alpha-, beta- and gamma-thrombin, respectively) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C). The affinity of DAPP, TAPB and TAPP for alpha- and beta-thrombin is higher than that found for benzamidine association around neutrality, converging in the acidic pH limb; in contrast, benzamidine, DAPP, TAPB and TAPP show the same value of the association inhibition constant (Ki; M-1) for gamma-thrombin over the whole pH range explored. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to alpha-, beta- and gamma-thrombin, as well as for DAPP, TAPB and TAPP association to gamma-thrombin reflects the acidic-pK shift, upon inhibitor binding of a single ionizing group. On the other hand, values of Ki for DAPP, TAPB and TAPP binding to alpha- and beta-thrombin appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues over the same pH range. By considering molecular models of the serine proteinase:inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).