Comparison of pharmacokinetic parameters of a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate

PURPOSE: The alteration of the pharmacokinetic parameters of the polypeptide BBI through conjugation with palmitic acid was examined. METHODS: 125I-BBI or 125I-Pal-BBI was administered iv to 6 week old CF-1 mice at a dose of 3 mg/kg. The mice were sacrificed at 5, 10, 20, 60, 120, 240, 360, and 480 min and the total radioactivity was determined for blood and each organ. The blood was analyzed on a Sephadex G-50 size-exclusion column to determine the amount of intact polypeptide present in the blood. From the amount of intact polypeptide at each time point, the pharmacokinetic parameters were determined. RESULTS: By conjugating three palmitic acids to each BBI molecule, the area under the curve (AUC) and mean residence time (MRT) increase by a factor of 10.8 and 2.8, respectively. There was also a difference in the organ distribution between the two treatments; while 125I-BBI was rapidly cleared from the kidneys, 125I-Pal-BBI was predominantly to the liver. Subsequent studies suggested that the binding of the conjugate to non-albumin serum proteins was most likely the cause of the altered pharmacokinetics. CONCLUSIONS: The residence time in the blood and the lipophilicity of BBI were increased upon conjugation with palmitic acid through a reversible disulfide linkage. Pharmacokinetic studies showed an increase in the AUC and a decrease in kidney clearance in palmitic acid conjugates, indicating a potential increase of the therapeutic efficacy of the polypeptide drug.     

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.     

Studies on the synthesis of proteinase inhibitors. II. Synthesis of cyclic nonapeptide fragments and analogs related to the reactive sites of soybean Bowman-Birk inhibitor

Several heterodetic cyclic nonapeptides modeled after the disulfide loops of Bowman-Birk inhibitor which involve either the anti-tryptic or anti-chymotryptic region have been synthesized by the conventional method. The inhibitory properties and the stabilities of these peptides toward trypsin and chymotrypsin, as well as the properties of dimers of the above nonapeptides, were examined.     

Conjugation of classic Bowman-Birk soy inhibitor with a copolymer of ethylene oxide and propylene oxide

A classical soybean inhibitor of the Bowman-Birk type (BBI) with a copolymer of ethylene oxide and propylene oxide (PE) has been synthesized. The BBI-PE conjugate contain five covalently bound polymeric chains per one protein molecule and retains its capacity to inhibit trypsin (Ki = 10(-10) M), alpha-chymotrypsin (Ki = 7 x 10(-8) M) and human granulocyte elastase (Ki = 3 x 10(-8) M). The preservation of the antiproteinase activity in the antichymotrypsin center creates a prerequisite for the manifestation of the anticarcinogenic effect of the inhibitor.     

Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitors

A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural L-amino acids (except for cysteine), plus the non-natural L-amino acids ornithine and norleucine, The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN', alpha-chymotrypsin, trypsin, anhydro-alpha-chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to alpha-chymotrypsin, two of which also bind to anhydro-alpha-chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K(i)) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K(i) values. In the case of the alpha-chymotrypsin inhibitors, the peptides with the lowest K(i) (0.1-0.05 mM) were the only peptides that bound to anhydro-alpha-chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that alpha-chymotrypsin and trypsin are inhibited by these cyclic peptides.     

Isolation of a protein Z-dependent plasma protease inhibitor

Human protein Z (PZ) is a 62,000-Mr, vitamin K-dependent plasma protein whose structure is similar to coagulation factors VII, IX, X, protein C, and protein S, but whose function is not known. The procoagulant activity of factor Xa in a one-stage plasma coagulation assay is reduced when factor Xa is first incubated with PZ. This apparent inhibitory effect is time dependent, requires the presence of calcium ions and procoagulant phospholipids (rabbit brain cephalin), and appears predominantly related to the incubation period of PZ with cephalin. In serum the initial rate of inhibition of factor Xa with calcium ions and cephalin also is enhanced in the presence PZ. A PZ-dependent protease inhibitor (ZPI) has been isolated from plasma. ZPI is a 72,000-Mr single-chain protein with an N-terminal amino acid sequence of LAPSPQSPEXXA (X = indeterminate) and an estimated concentration in citrate-treated plasma of 1.0-1.6 microg/ml. In systems using purified components, the factor Xa inhibition produced by ZPI is rapid (>95% within 1 min by coagulation assay) and requires the presence of PZ, calcium ions, and cephalin. The inhibitory process appears to involve the formation of a factor Xa-PZ-ZPI complex at the phospholipid surface.     

Molecular phylogenetic characterization of Streptomyces protease inhibitor family

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them "SSI-like proteins" (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.     

Combination of serine protease inhibitor FUT-175 and thromboxane synthetase inhibitor OKY-046 decreases cerebral vasospasm in patients with subarachnoid hemorrhage

The preventive effect of the serine protease inhibitor FUT-175 (nafamostat mesilate), a potent inhibitor of the complement system, against vasospasm was evaluated in 34 high risk patients with thick and diffuse subarachnoid hemorrhage (SAH) demonstrated by computed tomography corresponding to Fisher group 3. All patients underwent surgery within 96 hours following SAH and received the thromboxane A2 synthetase inhibitor, OKY-046, as part of standard care. FUT-175 (40-160 mg/day) was administered during the initial 4 days following surgery. 455 patients treated without FUT-175 in the Nagasaki SAH Data Bank (non-FUT group) formed the control group. FUT-175 significantly decreased the incidence of symptomatic vasospasm in patients with severe neurological grade (Hunt and Hess grade 3, p < 0.02; Hunt and Hess grade 4, p < 0.02). The incidence of favorable outcome was 76.5% in the FUT group and 60.4% in the non-FUT group, but not statistically different. However, when patients of Hunt and Hess grade 5 were excluded, the FUT group had a significantly improved outcome (p < 0.05). This study suggests that FUT-175 has an additive effect to OKY-046 in preventing vasospasm in high risk patients with severe SAH.     

The ex vivo human placental transfer of the anti-HIV nucleoside inhibitor abacavir and the protease inhibitor amprenavir

OBJECTIVE: The transfer of abacavir, a new nucleoside inhibitor, and amprenavir, a new protease inhibitor, used for the treatment of human immunodeficiency virus, has been studied in the ex vivo human placental model. METHODS: The ex vivo human placental model used C14 antipyrine to determine the transport fraction and clearance index of these compounds at both the peak and trough serum concentrations. The clearance index accumulation and tissue concentrations were determined for each drug by high pressure liquid chromatography. RESULTS: The clearance index of abacavir was 0.47 +/- 0.19 and 0.50 +/- 0.07 at peak and trough concentrations, respectively. The clearance index of amprenavir was 0.38 +/- 0.09 and 0.14 +/- 0.08 at peak and trough concentrations, respectively. There was no unusual accumulation of either drug in the media or tissue when the perfusion system was closed. CONCLUSION: Abacavir is the first nucleoside compound studied in the perfusion system with a high clearance index. The transfer of the protease inhibitor amprenavir had a clearance index 2.75 times greater than the clearance index of ritonavir at peak concentration determined in a previous study. At trough concentration the clearance index was much less than at the peak concentration. A similar result was found with ritonavir.     

Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor

Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-kappaB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417-426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins-interleukin-10 (IL-10) and IL-6-also induced SLPI, while TNF and IL-1beta did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules-taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls-also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI's slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria.     

Cysteine proteases of Trichomonas vaginalis degrade secretory leukocyte protease inhibitor

Loss of heterozygosity (LOH) of chromosomal arm 8p has been reported to occur at high frequency for a number of common forms of human cancer, including breast cancer. The objectives of this study were to define the regions on this chromosomal arm that are likely to contain breast cancer tumor suppressor genes and to determine when loss of chromosomal arm 8p occurs during breast cancer progression. For mapping the tumor suppressor gene loci, we evaluated 60 cases of infiltrating ductal cancer for allelic loss using 14 microsatellite markers mapped to this chromosomal arm and found LOH of 8p in 36 (60%) of the tumors. Whereas most of these tumors had allelic loss at all informative markers, five tumors had partial loss of 8p affecting two nonoverlapping regions. LOH for all but one of the tumors with 8p loss involved the region between markers D8S560 and D8S518 at 8p21.3-p23.3, suggesting that this is the locus of a breast cancer tumor suppressor gene. We then studied LOH of 8p in 38 cases of ductal carcinoma in situ (DCIS) with multiple individually microdissected tumor foci evaluated for each case. LOH of 8p was found in 14 of the DCIS cases (36%), including 6 of 16 cases of low histological grade and 8 of 22 cases of intermediate or high histological grade. In four of these DCIS cases, 8p LOH was seen in some but not all of the multiple tumor foci examined. These data suggest that during the evolution of these tumors, LOH of 8p occurred after loss of other chromosomal arms that were lost in all tumor foci. Thus, LOH of 8p, particularly 8p21.3-p23, is a common genetic alteration in infiltrating and in situ breast cancer. Although 8p LOH is common even in low histological grade DCIS, this allelic loss often appears to be preceded by loss of other alleles in the evolution of breast cancer.     

Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation

Protease nexin-1 (PN-1), a member of the serpin superfamily, controls the activity of extracellular serine proteases and is expressed in the brain. Mutant mice overexpressing PN-1 in brain under the control of the Thy-1 promoter (Thy 1/PN-1) or lacking PN-1 (PN-1-/-) were found to develop epileptic activity in vivo and in vitro. Theta burst-induced long-term potentiation (LTP) and NMDA receptor-mediated synaptic transmission in the CA1 field of hippocampal slices were augmented in Thy 1/PN-1 mice and reduced in PN-1-/- mice. Compensatory changes in GABA-mediated inhibition in Thy 1/PN-1 mice suggest that altered brain PN-1 levels lead to an imbalance between excitatory and inhibitory synaptic transmission.     

Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy

The structure-activity studies leading to the potent and clinically efficacious HIV protease inhibitor ritonavir are described. Beginning with the moderately potent and orally bioavailable inhibitor A-80987, systematic investigation of peripheral (P3 and P2') heterocyclic groups designed to decrease the rate of hepatic metabolism provided analogues with improved pharmacokinetic properties after oral dosing in rats. Replacement of pyridyl groups with thiazoles provided increased chemical stability toward oxidation while maintaining sufficient aqueous solubility for oral absorption. Optimization of hydrophobic interactions with the HIV protease active site produced ritonavir, with excellent in vitro potency (EC50 = 0.02 microM) and high and sustained plasma concentrations after oral administration in four species. Details of the discovery and preclinical development of ritonavir are described.