The effect on serum lipids and oxidized low-density lipoprotein of supplementing self-selected low-fat diets with soluble-fiber, soy, and vegetable protein foods.

An increased intake of soluble fiber and soy protein may improve the blood lipid profile. To assess any additional benefit on serum lipids of providing soy protein and soluble-fiber foods to hyperlipidemic subjects already consuming low-fat, low-cholesterol therapeutic diets, 20 hyperlipidemic men and postmenopausal women completed 8-week test and control dietary treatments in a randomized crossover design as part of an ad libitum National Cholesterol Education Program (NCEP) step 2 therapeutic diet (<7% saturated fat and <200 mg/d cholesterol). During the test phase, foods high in soy, other vegetable proteins, and soluble fiber were provided. During the control phase, low-fat dairy and low-soluble-fiber foods were provided. Fasting blood lipid and apolipoprotein levels were measured at 4 and 8 weeks of each phase. On the test diet, 12 +/- 2 g/d soy protein was selected from the foods chosen. Direct comparison of test and control treatments indicated an elevated high-density lipoprotein (HDL) cholesterol concentration on the test diet (6.4% +/- 2.4%, P = .013) and a significantly reduced total to HDL cholesterol ratio (-5.9% +/- 2.3%, P = .020). The proportion of conjugated dienes in the low-density lipoprotein (LDL) cholesterol fraction was significantly reduced (8.5% +/- 3.3%, P = .020) as a marker of oxidized LDL. A combination of acceptable amounts of soy, vegetable protein, and soluble-fiber foods as part of a conventional low-fat, low-cholesterol therapeutic diet is effective in further reducing serum lipid risk factors for cardiovascular disease.

Combined effect of vegetable protein (soy) and soluble fiber added to a standard cholesterol-lowering diet.

Dietary treatment of hyperlipidemia focuses on reducing saturated fat and dietary cholesterol. Other aspects of diet are not emphasized at present, despite growing evidence that a number of plant components decrease serum cholesterol. We therefore determined whether a combination of two plant components, vegetable protein and soluble fiber, further reduce serum lipids when incorporated into the currently advocated low-saturated-fat diet. Thirty-one hyperlipidemic men and women ate two 1-month low-fat (<7% of total energy from saturated fat), low-cholesterol (<80 mg cholesterol/d) metabolic diets in a randomized crossover study. The major differences between test and control diets were an increased amount of vegetable protein (93% v 23% of total protein), of which 33 g/d was soy, and a doubling of soluble fiber. Fasting blood samples were obtained at the start and end of each phase. On the last 3 days of each phase, fecal collections were obtained. Compared with the low-fat control diet, the test diet decreased total cholesterol (6.2% +/- 1.2%, P < .001), low-density lipoprotein (LDL) cholesterol (6.7% +/- 1.7%, P < .001), apolipoprotein B (8.2% +/- 1.2%, P < .001), and the ratios of LDL to high-density lipoprotein (HDL) cholesterol (6.3% +/- 2.0%, P = .004) and apolipoprotein B to A-I (5.4% +/- 1.5%, P = .001). A combination of vegetable protein and soluble fiber significantly improved the lipid-lowering effect of a low-saturated-fat diet. The results support expanding the current dietary advice to include increased vegetable protein and soluble fiber intake so that the gap in effectiveness between a good diet and drug therapy is reduced.

Health aspects of partially defatted flaxseed, including effects on serum lipids, oxidative measures, and ex vivo androgen and progestin activity: a controlled crossover trial.

BACKGROUND: Currently there is considerable interest in the potential health benefits of oil seeds, such as soy and flaxseed, especially in relation to cardiovascular disease and cancer. OBJECTIVE: We therefore evaluated health aspects of partially defatted flaxseed in relation to serum lipids, indicators of oxidative stress, and ex vivo sex hormone activities. DESIGN: Twenty-nine hyperlipidemic subjects (22 men and 7 postmenopausal women) completed two 3-wk treatment periods in a randomized, crossover trial. Subjects were given muffins that contributed approximately 20 g fiber/d from either flaxseed (approximately 50 g partially defatted flaxseed/d) or wheat bran (control) while they consumed self-selected National Cholesterol Education Program Step II diets. Both muffins had similar macronutrient profiles. Treatment phases were separated by > or = 2 wk. RESULTS: Partially defatted flaxseed reduced total cholesterol (4.6+/-1.2%; P = 0.001), LDL cholesterol (7.6+/-1.8%; P < 0.001), apolipoprotein B (5.4+/-1.4%; P = 0.001), and apolipoprotein A-I (5.8+/-1.9%; P = 0.005), but had no effect on serum lipoprotein ratios at week 3 compared with the control. There were no significant effects on serum HDL cholesterol, serum protein carbonyl content, or ex vivo androgen or progestin activity after either treatment. Unexpectedly, serum protein thiol groups were significantly lower (10.8+/-3.6%; P = 0.007) at week 3 after the flaxseed treatment than after the control, suggesting increased oxidation. CONCLUSIONS: These data indicate that partially defatted flaxseed is effective in lowering LDL cholesterol. No effects on lipoprotein ratios, ex vivo serum androgen or progestin activity, or protein carbonyl content were observed. The significance of increased oxidation of protein thiol groups with flaxseed consumption requires further investigation.

N2- and C8-substituted oligodeoxynucleotides with enhanced thrombin inhibitory activity in vitro and in vivo.

2'-Deoxyguanosine (G) analogues carrying various hydrophobic substituents in the N2 and C8 positions were synthesized and introduced through solid-phase synthesis into 15-mer oligodeoxynucleotide, GGTTGGTGTGGTTGG, which forms a chairlike structure consisting of two G-tetrads and is a potent thrombin inhibitor. The effects of the substitutions at N2 and C8 of the G-tetrad-forming G residues on the thrombin inhibitory activity are relatively small, suggesting that these substitutions cause relatively small perturbations on the chairlike structure formed by the oligodeoxynucleotide. Introduction of a benzyl group into N2 of G6 and G11 and naphthylmethyl groups into N2 of G6 increased the thrombin inhibitory activity, whereas other substituents in these positions had almost no effect or decreased the activity. Particularly, the oligodeoxynucleotide carrying a 1-naphthylmethyl group in the N2 position of G6 showed an increase in activity by about 60% both in vitro and in vivo. Substitutions on the N2 position of other G residues had little effect or decreased the activity. Introduction of a relatively small group, such as methyl and propynyl, into the C8 positions of G1, G5, G10, and G14 increased the activity, presumably due to the stabilization of a chairlike structure, whereas introduction of a large substituent group, phenylethynyl, decreased the activity, probably due to the steric hindrance.

A novel oligodeoxynucleotide inhibitor of thrombin. II. Pharmacokinetics in the cynomolgus monkey.

PURPOSE: To determine the pharmacokinetics of GS-522, an oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, after constant infusion and bolus administration in the cynomolgus monkey. METHODS: Using a stability indicating HPLC method, the GS-522 plasma concentration versus time data were obtained after constant infusion (0.1, 0.3, 0.5 mg/kg/min) and bolus administration (11.25 and 22.5 mg/kg). Plasma data after bolus administration was fit to a three-compartment model. RESULTS: The half-lives for the alpha and beta phases were 1.4 and 5.4 min, respectively. Steady state GS-522 concentrations were reached within 10 minutes after initiation of constant infusions. Termination of infusions resulted in a rapid elimination of GS-522 with an average elimination half-life equal to 1.5 min. The Vss calculated from both the constant infusion and bolus data approximated the blood volume of the monkey. Substitution of the phosphodiester backbone at the 3' end of GS-522 with two phosphorothioate linkages did not substantially effect the elimination half-life upon termination of infusion. CONCLUSIONS: These data in conjunction with published biodistribution data suggest that oligodeoxynucleotides are rapidly cleared from plasma by tissue uptake and that little efflux back into blood takes place. Additionally, strategies designed to increase oligodeoxynucleotide resistance to exonucleases will not dramatically increase plasma half-lives.

Selection of a suppressor mutation that restores affinity of an oligonucleotide inhibitor for thrombin using in vitro genetics.

The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite I. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer. In contrast, aptamers selected against R70E thrombin were able to bind and inhibit both wild-type and R70E thrombins, and displayed potassium-independent inhibition. Aptamers selected against R70-E thrombin bound to sites identical or overlapping with that of the prototype thrombin aptamer. These aptamers retained the potential to form double G-tetrad structures; however, these structures would be destabilized by a T-->A substitution, disrupting the T4-T13 base pairing found in the prototype. This destabilization appeared to be partially compensated by newly recruited structural elements. Thus, selection against R70E thrombin did not lead to aptamers that bound to alternative sites, but instead to ssDNA structures with a suppressor mutation that accommodated the mutation in thrombin within a double G-tetrad context. These results provide insight into the aptamer-thrombin interaction and suggest that the binding site for the prototype is the dominant aptamorigenic site on thrombin.

Pilot study of the efficacy of a thrombin inhibitor for use during cardiopulmonary bypass.

Heparin is normally used for anticoagulation during cardiopulmonary bypass (CPB), but its use is contraindicated in patients with a history of heparin-induced thrombocytopenia, heparin-provoked thrombosis, or both. Heparin therapy can also be ineffective due to heparin resistance. A short-acting, oligonucleotide-based thrombin inhibitor (thrombin aptamer) may potentially serve as a substitute for heparin in these and other clinical situations. We tested a novel thrombin aptamer in a canine CPB pilot study to determine its anticoagulant efficacy, the resultant changes in coagulation variables, and the aptamer's clearance mechanisms and pharmacokinetics. Seven dogs were studied initially: Four received varied doses of the aptamer (to establish the pharmacokinetic profile) and 3 received heparin. Subsequently, 4 other dogs underwent CPB, receiving a constant infusion of the aptamer before CPB (to characterize the baseline coagulation status), with partial CPB and hemodilution, during 60 minutes of total CPB, and, finally, after a 2-hour recovery period. At a 0.5 mg.kg-1.min-1 dose, the activated clotting time rose with aptamer infusion from 106 +/- 12 seconds to 187 +/- 8 seconds (+/- 1 standard deviation) (p = 0.014), increased further with hemodilution (to 259 +/- 41 seconds; p = 0.017), and was even more prolonged during total CPB (> 1,500 seconds; p < 0.001). This later increase in the activated clotting time paralleled a rise in the plasma concentration of the thrombin aptamer during total CPB, as determined by high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

The discovery and characterization of a novel nucleotide-based thrombin inhibitor.

Thrombin is a serine protease that plays a pivotal role in thrombosis and hemostasis, and is a major target for anticoagulation and cardiovascular disease therapy. Using a novel in vitro selection/amplification technique, we have identified a new class of thrombin inhibitors based on single-stranded DNA (ssDNA) oligodeoxyribonucleotides (oligos). These thrombin inhibitors are the first example of the use of this technique to obtain ssDNA oligos that bind a target protein that does not interact physiologically with nucleic acid. Here, we review how iterative selection and amplification were used to identify short ssDNA sequences that bind and inhibit thrombin (Bock et al., Nature 355 (1992) 564-566), and the tertiary structure of one aptamer sequence (Wang et al., Biochemistry 32 (1993) 1899-1904). Results from in vitro and in vivo studies are also summarized (Griffin et al., Blood 81 (1993) 3271-3276). The discovery of a new class of thrombin inhibitors using this technology demonstrates the power of this new approach for rapid drug discovery and development.

The single-stranded DNA aptamer-binding site of human thrombin.

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.

In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits.

Using a novel in vitro selection/amplification technique, we have recently identified a new class of thrombin inhibitors based on single-stranded DNA oligonucleotides. One oligonucleotide, GGTTGGTGTGGTTGG (thrombin, aptamer), showed potent anticoagulant activity in vitro. We have initiated pharmacologic studies in cynomolgus monkeys to study the thrombin aptamer's in vivo anticoagulant properties. Upon infusion of the thrombin aptamer, anticoagulation was rapidly achieved, with a plateau reached within 10 minutes. There was a linear dose-response relationship between thrombin aptamer infusion rate and prolongation of plasma prothrombin time. Ten minutes after the infusion was stopped, no prolongation of prothrombin time was observed, indicating that the thrombin aptamer has an extremely short in vivo half-life, estimated to be 108 +/- 14 seconds. In addition, inhibition of thrombin-induced platelet aggregation in platelet-rich plasma was observed ex vivo without an effect on collagen-induced aggregation, indicating that the inhibition was specific for thrombin and not due to a nonspecific inhibitory effect on platelets. To exploit the short in vivo half-life of the thrombin aptamer, its ability to achieve regional anticoagulation in an extracorporeal hemofiltration circuit in sheep was tested. Doubling of the prothrombin time in the circuit was observed, whereas the systemic prothrombin time was minimally prolonged. We conclude that the thrombin aptamer is a potent anticoagulant in vivo, and specifically inhibits thrombin-induced platelet aggregation ex vivo. The rapid onset of action and short half-life in vivo suggest that the thrombin aptamer may be useful in anticoagulation with extracorporeal circuits and may have distinct advantages in certain acute clinical settings.

Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving.

Nearest neighbor interactions affect the stabilities of triple-helical complexes. Within a pyrimidine triple-helical motif, the relative stabilities of natural base triplets T.AT, C + GC, and G.TA, as well as triplets, D3.TA and D3.CG, containing the nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3) were characterized by the affinity cleaving method in the context of different flanking triplets (T.AT, T.AT: T.AT, C + GC: C + GC, T.AT: G + GC, C + GC). The to be insensitive to substitutions in either the 3' or 5' directions, while the relative stabilities of triple helices containing C + GC triplets decreased as the number of adjacent C + GC triplets increased. Triple helices incorporating a G.TA interaction were most stable when this triplet was flanked by two T.AT triplets and were adversely affected when a C + GC triplet was placed in the adjacent 5' direction. Similarly, complexes containing D3.TA or D3.CG triplets were most stable when the triplet was flanked by two T.AT triplets but were destabilized when the adjacent 3' neighbor position was occupied with a C + GC triplet. This information regarding sequence composition effects in triple-helix formation establishes a set of guidelines for targeting sequences of double-helical DNA by the pyrimidine triple-helix motif.

Selection of single-stranded DNA molecules that bind and inhibit human thrombin.

Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.

Recognition of thymine adenine.base pairs by guanine in a pyrimidine triple helix motif.

Oligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double-helical DNA. In the pyrimidine-Hoogsteen model, a binding size of greater than 15 homopurine base pairs affords greater than 30 discrete sequence-specific hydrogen bonds to duplex DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts, it is desirable to determine whether oligonucleotides can be used to bind all four base pairs of DNA. A general solution would allow targeting of oligonucleotides (or their analogs) to any given sequence in the human genome. A study of 20 base triplets reveals that the triple helix can be extended from homopurine to mixed sequences. Guanine contained within a pyrimidine oligonucleotide specifically recognizes thymine.adenine base pairs in duplex DNA. Such specificity allows binding at mixed sites in DNA from simian virus 40 and human immunodeficiency virus.