Membrane-protease interactions. III: A consideration of the difference in binding potential of pancreatic proteases to erythrocytes and erythrocyte ghosts.

Trypsin and chymotrypsin readily bind to human erythrocyte ghosts and to resealed right-side-out ghosts, but not to intact erythrocytes, as followed with [3H]trypsin and [3H]chymotrypsin and with cold proteases in a caseinolytic assay. The proteases freely reacted with casein in the presence of intact cells. Trypsin activated trypsinogen over an 8-hr time course at a faster rate in the presence of erythrocytes than in the absence thereof, after a slight initial delay. Trypsinogen did not bind to intact erythrocytes, thereby behaving comparably to trypsin. These results suggest that different microenvironments exist about the erythrocyte ghosts and the intact erythrocytes, thereby permitting the proteases to bind to the former but not to the latter. Hence, in the absence of considerable ghosts in circulating blood, which may mask the binding site of the proteases, the proteases may be more readily accessible for interaction with circulating serpins, leading to inactivation of the proteases and protection from their degradative potential. The presence of the serpins in circulating blood may assist in the control of the degradative power of the pancreatic proteases in pancreatitis and may negatively modulate such processes as thrombosis, activation of the complement system, and vascular remodeling.

Antithrombotic activity of recombinant tick anticoagulant peptide and heparin in a rabbit model of venous thrombosis.

An in vivo rabbit model of venous thrombosis which includes physiological blood flow was used to compare the efficacy of the potent and specific factor Xa inhibitor recombinant tick anticoagulant peptide (rTAP) with standard heparin in the prevention of venous thrombus formation. In anesthetized rabbits, an autologous thrombus was induced with thrombin in a jugular vein and the increase in thrombus size was determined by measuring the accretion of intravenously injected [125I]fibrin(ogen) onto the developing thrombus. The effects of rTAP on hemostasis were monitored by changes in APTT values and template bleeding times. Inhibition of thrombus formation by an intravenous bolus followed by infusion of either rTAP or heparin exhibited a dose-response relationship with an IC50 of 0.9 micrograms/kg/min and 0.12 units/kg/min, respectively. At the IC50 doses, both rTAP and heparin inhibited fibrin(ogen) deposition without any significant effect on APTT or bleeding times. Bleeding times were modestly elevated at the fully efficacious doses of rTAP and heparin. Significant changes in APTT (1.9 +/- 0.3 fold over baseline) were only evident at the highest dose of rTAP while heparin caused a significant dose-dependent increase from 1.3 +/- 0.2 to greater than 4.2 +/- 0.6 fold over baseline. Therefore, in this rabbit model of venous thrombosis, specific inhibition of factor Xa by rTAP is an effective antithrombotic mechanism that does not require changes in systemic hemostatic parameters.

A single administration of LFA-1 antibody confers prolonged allograft survival.

C57BL/6 (B6) thyroid gland transplanted to the left kidney capsule of an allogeneic (BALB/c) host was typically rejected in 14 days. A single administration of 500 micrograms of an antibody to the adhesion molecule, leucocyte function-associated antigen (LFA-1, CD11a), prevented all thyroid allograft rejection for at least 70 days. Fifty percent of the treated recipients retained intact allografts for 470 days. However, the same treatment with anti-CD11a could not protect a sensitized BALB/c mouse from rejecting a second B6 thyroid allograft. Production of donor-specific alloantibodies elicited by allograft rejection was also inhibited in this system. In this transplant model, the Ab therapy is more efficacious than that of FK506, administered daily for 14 days at 15 mg/kg. These results demonstrate the remarkable effect of an anti-LFA-1 antibody in promotion of allograft survival.

The effects of age and race on nitrendipine pharmacokinetics and pharmacodynamics.

The pharmacokinetics and pharmacodynamics of a single 20-mg dose of nitrendipine (NTP) were studied in four groups of subjects (n = 9 per group). Group 1 were young white normotensive males, Group 2 were elderly white hypertensive males, Group 3 were black hypertensive males aged 40-55 years, and Group 4 were white hypertensive males aged 40-55 years. All other medications were withdrawn prior to NTP dosing. NTP was given in the morning 1 h after breakfast. Plasma samples for NTP assay were collected at predetermined times up to 48 h after dosing. Blood pressure was monitored before dosing, and at 0.5, 1, 3, 5, 12, and 24 h postdose. Pharmacokinetic parameters were found to be dependent on age. The area under the curve for Groups 1, 2, 3, and 4 was 50.5 +/- 19.4, 186 +/- 120, 107 +/- 49, and 88 +/- 43 ng h/ml, respectively (p less than 0.05). Corresponding values of elimination half-life were 9.9 +/- 1.3, 20 +/- 6.5, 13.3 +/- 6.1, and 15.9 +/- 8.0 h (p less than 0.05). Both diastolic and systolic blood pressures were significantly reduced from the baseline value in Groups 2, 3, and 4, with diastolic pressure remaining significantly lower than baseline at 24 h postdose. Based on the increased plasma levels and slower elimination of NTP in the elderly, as well as the measured blood pressure lowering, once daily dosing of NTP may be appropriate in some patients.

Bioequivalence and metabolism of nitrendipine administered orally to healthy volunteers.

Relative bioavailability of 5-, 10-, and 20-mg nitrendipine tablets was determined in a four-way crossover bioequivalence study involving 22 normal male volunteers. Liquid suspension of nitrendipine was used as a reference. Plasma and urine samples collected during each study period were assayed by high performance liquid chromatographic and capillary gas chromatographic (GC) procedures for nitrendipine and the nitrendipine pyridine metabolite. Four other more polar urinary nitrendipine metabolites were also analyzed in urine by the GC procedure, which involved diazomethane esterification. Although relative bioavailability of the tablets ranged from 58.0 to 69.9%, there was no statistically significant difference in the area under the curve among the doses. Since 35 to 43% of both the liquid and tablet doses was recovered in the urine of volunteers, excretion of urinary metabolites appears to be independent of the dosage form. However, a rank-order correlation between the relative tablet bioavailability and cumulative amounts of excreted metabolites was observed. Nitrendipine, its pyridine metabolite, and the glucuronide conjugates were also detected in the urine, but the amount of nitrendipine and its pyridine metabolite did not exceed 0.1% of dose, whereas the glucuronides accounted for about 8% of the dose.

Steady-state pharmacokinetics of nitrendipine in hepatic insufficiency.

As a calcium antagonist, nitrendipine will be used in the treatment of various diseases in patients with hepatic insufficiency, and it is important to know if they require modified dosing schedules. In this study, six patients with biopsy-confirmed cirrhosis and six age/sex-matched normal healthy subjects were given 10 mg nitrendipine as a single dose on day 1 and 10 mg nitrendipine every 12 h from day 3 through the first dose on day 8. Blood levels of nitrendipine were determined to confirm the attainment of steady state and evaluate the pharmacokinetics in each group. Nitrendipine concentrations were consistently higher in the hepatic group. On day 1, the maximum concentration (Cmax) in the normals was 4.67 +/- 2.60 ng/ml and in the hepatic patients 16.87 +/- 9.42 ng/ml. On day 8, these values were 8.60 +/- 8.82 ng/ml and 27.37 +/- 8.56 ng/ml, respectively. The time to Cmax was not significantly different in the two groups. The elimination half-life was only slightly prolonged from 15.29 +/- 7.25 h in the normals to 19.57 +/- 4.28 h in the hepatic impairment group. This resulted in a marked increase in the area under the concentration-time curve from 28.71 +/- 28.92 ng . h/ml for the normals to 119.56 +/- 34.39 ng . h/ml for the hepatic patients on day 1 and similar results on day 8. Trough levels at steady state were expectedly higher in the hepatic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell cycle analysis of cultured mammalian cells after exposure to 4,5',8-trimethylpsoralen and long-wave ultraviolet light.

Cell cycle analysis was used to study the the effect of 4,5'8-trimethylpsoralen (TMP) and long-wave ultraviolet light (UV-A) on cultured mammalian cells. DNA distribution patterns were measured for murine melanoma cells (a cloned line of Cloudman S91) and a strain of diploid human skin fibroblasts (CRL 1295) using both a microfluorimetry procedure and flow cytometry. The untreated cells and those receiving TMP along and UV-A alone had identical DNA content as assessed at several posttreatment intervals (0-72 hr). The majority of cells in control groups contained a G1 DNA content, whereas exposure to TMP (2 x 10(-7) M) plus UV-A (1 Joule/cm2) led to the accumulation of cells in the G2 phase. These observations were similar for each cell type and both analytical techniques were in excellent agreement. The finding that psoralen plus UV-A induces a phase-specific G2 blockade in cultured cells has important implications for understanding the mechanisms which account for enhanced pigmentation and suppression of cellular proliferation following exposure to these agents in vivo.

Comparative pharmacologic effects on tissue factor activity in normal cells and an established cell line.

The tissue factor (thromboplastin) activity of cells grown in vitro is modulated by exogenous drugs. The activity of human foreskin fibroblasts and umbilical vein smooth muscle cells is enhanced by 10(-6) M hydrocortisone or 1 mM butyrate. Activity is suppressed in these cells by 10(-6) M colchicine whereas 10(-4) M chloroquine has little or no effect. Two established cell lines, WISH or HeLa cells, have elevated tissue factor activity in the presence of colchicine or chloroquine and suppressed activity with exogenous hydrocortisone. Their activity is also decreased by 10 mM butyrate whereas 1 mM butyrate does not alter activity. Colchicine and butyrate apparently act via a mechanism unrelated to their effect on microtubules since it is possible to dissociate activity changes from morphologic changes. Umbilical vein endothelial cell tissue factor activity responds uniquely to exogenous drugs. Hydrocortisone or 10(-5) M vinblastine (or colchicine) only minimally alters activity. Endothelial cells are not simply refractory toward all drugs, however, since chloroquine dramatically enhances activity whereas 1 mM butyrate suppresses it. The low specific activity of endothelial cells and their apparently unique drug response may be another measure of their function as an in vivo hemostatic barrier.

The hydrolysis of biologically active peptides by bovine lung tissue factor (thromboplastin).

Tissue factor apoprotein and relipidated tissue factor preparations extensively hydrolyze bradykinin, Lys-bradykinin, Met-Lys-bradykinin, substance P, [Asp1, Ile5]-angiotensin II, [Asp1, Ile5]-angiotensin I, and human fibrinopeptide A while acting more slowly on [Sar1, Ile5]-angiotensin II, [Me2Gly1, Ile5]-angiotensin II, bradykinin potentiating pentapeptide from B. jararaca, luteinizing hormone-releasing hormone, melanocyte stimulating hormone-release-inhibiting factor (Pro-Leu-Gly-NH2), and oxytocin. No hydrolysis of thyrotropin-releasing factor or bradykinin potentiating nonapeptide from B. jararaca is observed. Relipidated and apoprotein tissue factor act at identical rates under the conditions of the assay. Dansylation and chromatography of tissue factor-peptide incubation mixtures further indicate that relipidated and apoprotein tissue factor also hydrolyze peptides by identical mechanisms. No fewer than six bonds are hydrolyzed in bradykinin while the angiotensins and substance P are degraded to constituent amino acids. Only the N-terminal alanine is released from fibrinopeptide A. 2-Mercaptoethanol greatly inhibits the hydrolysis of bradykinin by relipidated tissue factor.