1802

References (/) TAYLOR CW, ALBERTS DS, KETCHAM MA,

ET AL: Clinical pharmacology of a novel diarylsulfonylurea anticancer agent. J Clin Oncol 7:1733-1740, 1989 (2) HAINSWORTH JD, HANDE KR, SATTERLEE

WG, ET AL: Phase I clinical study of N[(4-chlorophenyl)amino]carbonyl-2,3-dihydro-l //-indene-5-sulfonamide (LY186641). Cancer Res 49:5217-5220, 1989 (3) HOWBERT

JJ,

GROSSMAN

CS, CROWELL

TA, ET AL: Novel agents effective against solid tumors: The diarylsulfonylureas. Synthesis, activities and analysis of quantitative structure-activity relationships. J Med Chem 33:2393-2407, 1990

Plasminogen Activator and Its Inhibitor in Cancer Patients Treated With Tumor Necrosis Factor Theodore F. Logan,* Mohamed A. Virji, William E. Gooding, Franklin A. Bontempo, Marc S. Ernstoff, John M. Kirkwood

(4) HOUGHTON PJ, BAILEY FC, GERMAIN GS,

ET AL: A'-(5-indanylsulfonyl)-Af'-(4-chlorophenyljurea, a novel agent equally cytotoxic to nonproliferating human colon adenocarcinoma cells. Cancer Res 50:318-322, 1990 (5) HOUGHTON PJ, BAILEY FC, GERMAIN GS,

ET AL: Studies on the cellular pharmacology of yV-(4-methylphenylsulfonyl)-/v"-(4chlorophenyl)-urea. Biochem Pharmacol 39:1187-1192, 1990 (6) HOUGHTON PJ, BAILEY FC, HOUGHTON JA,

ET AL: Evidence for mitochondrial localization of A'-(4-methylphenylsulfonyl)-Af'-(4chlorophenyl)urea in human colon adenocarcinoma cells. Cancer Res 50:664-668, 1990 (7) HOUGHTON PJ, HOUGHTON JA, MYERS L,

ET AL: Evaluation of 7V-(5-indanylsulfonyl)-W-(4-chlorophenyl)-urea against xenografts of pediatric rhabdomyosarcoma. Cancer Chemother Pharmacol 25:84-88, 1989 (8) INTERNATIONAL FEDERATION OF GYNECOL-

OGY AND OBSTETRICS: Changes in definitions of clinical staging for carcinoma of the cervix and ovary. Am J Obstet Gynecol 156:263-264, 1987 (9) LAM FC, HUNG CT, PERRIER DG: Estima-

tion of variance for harmonic mean halflives. J Pharm Sci 74:229-231, 1985 (10) THIGPEN JT, BLESSING JA, VANCE RB, ET

AL: Chemotherapy in ovarian carcinoma: Present role and future prospects. Semin Oncol 16:58-65, 1989 (//)

MCGUIRE WP, ROWINSKY EK, ROSENSHEIN

NB, ET AL: Taxol: A unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med 111:273-279, 1989 (12) SMITH

RP,

OLSON

methemoglobinemia. 10:253-268, 1973

MV:

Drug-induced

Semin

Hematol

GIVE TO THE UNITED NEGRO COLLEGE FUND. A Mind Is A Terrible Thing To Waste.

Background: We noted the presence of plasma fibrin degradation products in patients treated with recombinant human tumor necrosis factor (TNF) in a phase I trial. Purpose: To further define this observation, we investigated the effects of TNF on the

Downloaded from http://jnci.oxfordjournals.org/ at OCLC on July 26, 2015

cancer or other investigational agents (70). For example, a study of taxol by McGuire et al. (77) showed a 30% overall response rate (95% confidence interval = 16%-44%) in 40 assessable patients with advanced ovarian cancer. The dose-limiting hematologic toxic effects we observed (anemia and methemoglobinemia) were possibly more severe because our patients had been heavily pretreated with cisplatin and other myelosuppressive drugs. The compensatory bone marrow response to the decreased red blood cell survival induced by sulofenur was possibly limited in our patients. The 5/2-day schedule resulted in fewer patients requiring red blood cell transfusion than the 14-day schedule (31% versus 78%) and less severe methemoglobinemia. However, 31% of patients treated with the 5/2-day schedule still required dose reductions. Thus, the toxic effects associated with sulofenur therapy may limit the ability to deliver dose-intensive therapy and to achieve plasma levels similar to those that were shown to be very active in preclinical models. The major metabolites of sulofenur in plasma are the keto and hydroxy derivatives. Our data show that both compounds have a prolonged t 1/2 and larger Vp than the parent compound. Neither metabolite is active in preclinical tumor models, but both are metabolized to p-chloroaniline (data on file, Eli Lilly and Co.), a known cause of acquired methemoglobinemia (72). Indeed, in our study, the correlations of metabolite concentrations with methemoglobin levels were higher than the correlation of sulofenur concentrations with methemoglobin levels. The metabolic conversion to p-chloroaniline is more efficient in humans than in mice; thus, the preclinical models may have underpredicted the toxicity of sulofenur in humans. The toxic effects of anemia and methemoglobinemia may limit the ultimate clinical utility of diarylsulfonylureas until less toxic derivatives with alternate metabolic pathways can be identified. However, because of their broad-spectrum antitumor activity in preclinical models, the diarylsulfonylureas remain an exciting new class of anticancer compounds, and further development of these drugs is warranted.

Received April 2, 1992; revised August 26, 1992; accepted September 15, 1992. Supported in part by a grant from Knoll Pharmaceuticals, Whippany, N.J. T. F. Logan is a recipient of American Cancer Society Career Development Award #90-214. T. F. Logan, M. S. Ernstoff, J. M. Kirkwood, Division of Medical Oncology, Department of Medicine, University of Pittsburgh, and Pittsburgh Cancer Institute, Pittsburgh, Pa. Present address: M. S. Ernstoff, DartmouthHitchcock Medical Center, Section of Hematology/Oncology, Department of Medicine, Hanover, N.H. M. A. Virji, Department of Pathology, University of Pittsburgh, and Pittsburgh Cancer Institute. W. E. Gooding, Pittsburgh Cancer Institute. F. A. Bontempo, Divisions of Medical Oncology and Hematology, Department of Medicine, University of Pittsburgh. Portions of these data have been reported in abstract form in Proc ASCO (8:195, 1989) and in Clin Res (39:436A, 1991). We thank Ronald B. Herberman, M.D., and Ronald H. Goldfarb, Ph.D., for useful comments. We thank Ms. Mary Jo Dimasi for her technical help as well as the house staff of the hospitals of the University of Pittsburgh and Cheryl Tompkins, R.N., and the nursing and support staff of Unit 12-3 of the Presbyterian University Hospital for superb care of these patients. We are grateful to P. Nadler, M.D., D. Wood, and L. Pugliese at Knoll Pharmaceuticals for their support of this phase I trial. We thank Ms. Marcia Schmitz for her excellent secretarial skills. 'Correspondence to: Theodore F. Logan, M.D., Division of Medical Oncology, Department of Medicine, and Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Montefiore University Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213.

Journal of the National Cancer Institute

Vol. 84, No. 23, December 2, 1992

part, responsible for antitumor effects of TNF in experimental animals and possibly in humans (52). Preliminary analysis of TNF-treated patients in a phase I trial at our institution revealed the presence of plasma fibrin degradation products. However, no decrease in fibrinogen was detected (33). The fibrinolytic system was evaluated in these patients to examine the basis of these findings.

Patients and Methods Patients In the phase 1 trial, 24 patients were treated. Nine had colorectal carcinoma, seven had nonsmall-cell bronchogenic carcinoma, four had melanomas, two had renal cell carcinoma, and two had head and neck cancer (one squamous cell cancer and one adenoid cystic cancer). Eligibility criteria included the following: (a) Eastern Cooperative Oncology Group performance status of 2 or less; (b) normal biochemical indices of renal, hepatic (unless heptatic involvement by tumor), and hematologic functions; and (c) written informed consent. Patients were considered to be ineligible for the following reasons: active infection, cytostatic or immunosuppressive therapy within the previous 4-6 weeks, or concurrent therapy with aspirin, nonsteroidal anti-inflammatory agents, or steroids. This study was approved by the University of Pittsburgh Institutional Review Board.

Downloaded from http://jnci.oxfordjournals.org/ at OCLC on July 26, 2015

fibrinolytic system in patients en- minogen activator was preceded by tered in the same trial. Methods: In granulocytopenia, which may indithe 14 patients studied, fibrinolytic cate an association between a proparameters were measured by ana- posed TNF-induced granulocytelyzing blood samples for tissue plas- endothelial interaction in vivo and minogen activator and inhibitor at 0, release of tissue-type plasminogen 1, 2, 4, 6, and 18-24 hours after activator. Implications: These findinitiation of TNF treatment. We used ings demonstrating the effects of a chromogenic substrate method to TNF on the fibrinolytic system can determine activity of plasminogen be analyzed further in experimental activator and its inhibitor and an systems to determine the implications enzyme-linked immunosorbent assay for use of this agent as a biological (ELISA) to determine levels of anti- response modifier in cancer therapy. gen (tissue-type plasminogen activa- [J Natl Cancer Inst 84:1802-1810, tor). Molecular weight was deter- 1992] mined by zymographic assay. Results: TNF treatment was associated with tissue-type plasminogen activator induction within 1 hour of TNF initiation. The plasminogen activator Tumor necrosis factor (TNF) was produced was consistent with tissue- described as the endogenous protein type plasminogen activator derived causing hemorrhagic necrosis of transfrom endothelium as evidenced by planted tumors in mice treated with molecular weight analysis and BCG and endotoxin (1). It has been ELISA. Moreover, induction of plas- evaluated as an anticancer agent in minogen activator inhibitor occurred tumor cell cultures (2-5), laboratory following the release of tissue-type animals (4,6-9), and humans (10-16). plasminogen activator, and our data TNF is thought to be a mediator of suggest a dose-response effect for effects induced by endotoxin (17-19). TNF. At high doses (i.e., 200 and 240 Endotoxin alters the vascular en(ig/m2), there was a more rapid and dothelium (20,21), causing increased prolonged release of plasminogen production of tissue plasminogen acactivator inhibitor, which had an tivator inhibitor and release of von inverse relationship with the level of Willebrand factor (vWF). In preclinical antigenic tissue-type plasminogen ac- studies, TNF has been found to simtivator. Zymographic analysis ilarly affect the vascular endothelium, showed urokinase-type plasminogen with direct antiproliferative effects (22) activator activity in 13 of 14 patients. and production of granulocyte-macroIn three patients, simultaneous meas- phage colony-stimulating factor (23), urements of white blood cells and interleukin-1 (24), procoagulant activity tissue-type plasminogen activator re- (25), and tissue factor (26), as well as vealed a temporal association be- downregulation of the protein C pathtween the TNF-associated rapid way (26) and the release of plasgranulocytopenia at 30 minutes after minogen activator inhibitor (27-29). TNF initiation and release of tissueTNF causes an increase in protype plasminogen activator antigen. coagulant (versus anticoagulant) influConclusions: The results suggest a ences in vivo and may be an important positive association between TNF and factor in causing hypercoagulable states rapid induction of plasminogen ac- (30). In murine models, TNF caused tivator activity that is consistent with fibrin deposition in tumor vasculature an endothelial product. It is possible within 30 minutes of injection, an that, at high doses, TNF may interact effect partially inhibited by antidirectly with vascular endothelium, coagulation with 4-hydroxycoumarin leading to rapid and prolonged pro- (57). Monoclonal antibody against TNF duction of plasminogen activator in- can also partially block hemorrhagic hibitor. There was a dose-response necrosis in the endotoxin-treated mouse effect between TNF and release of transplant model (17). TNF may protissue-type plasminogen activator. foundly affect the vascular enThe release of tissue-type plas- dothelium, and these effects may be, in

Recombinant Human TNF Recombinant human TNF (Knoll Pharmaceuticals) was given in 2-hour intravenous infusions (generally in the morning), initially as a single dose, followed by four courses of five daily (2hour) intravenous doses given every 3rd week at dose levels of 40, 80, 160, 200, and 240 p-g/rn2. escalating between patients. Specific activity of TNF was 9.6 X 106 U/mg protein (L929 cytotoxicity assay), and the endotoxin content was less than 5.0 endotoxin U/mg protein (limulus lysate assay).

Blood Counts Complete differential blood leukocyte and platelet counts were obtained at designated times by peripheral venipuncture or from central venous catheters as previously described (34).

Analyses for Plasminogen Activators and Inhibitors Analyses for plasminogen activators and inhibitors were performed on venous blood samples obtained from the patients. Several of these patients also had blood samples taken during subsequent cycles of TNF. Samples were obtained before treatment and at 1, 2, 4, 6, and

REPORTS

1803

The immunoassay for tissue-type plasminogen activator was performed using an enzyme-linked immunosorbent assay (EL1SA) with antihuman tissue-type plasminogen activator polyclonal antibodies (Imunbind-5 tissue-type plasminogen activator ELISA kit; product 122; American Diagnostica, Inc., Greenwich, Conn.). The amount of antigen in plasma was measured as nanograms of tissue-type plasminogen activator per milliliter (37-39). Plasminogen activator inhibitor activity in the plasma samples that were treated with acid to remove labile inhibitors of plasminogen activator was determined by back-titration after the addition of a fixed amount of tissue-type plasminogen activator to each sample (40,41). Residual plasminogen activator enzymatic activity was then determined by the chromogenic substrate method. The plasminogen activator inhibitor activity was measured as arbitrary units (AU), with each arbitrary unit representing inhibition of 1 IU of tissue-type plasminogen activator. A calibration curve for the assay was constructed by the addition of tissue-type plasminogen activator to aliquots of pooled normal human plasma to obtain a standard curve that was linear in the assay range of 0-80 AU/mL. The plasma samples were also analyzed by casein-plasminogen zymography for plasminogen activator activity after polyacrylamide gel electrophoresis (PAGE) to determine the molecular weight characteristics of the plasminogen activator activity. The sodium dodecyl sulfate

1804

administered TNF at four dose levels. Venous blood was sampled before TNF administration and then at 1, 2, 4, 6, and 24 hours after infusion. Specifically, 13 samples were obtained before TNF, between 10 and 13 samples were obtained at 1-6 hours after treatment, and seven samples were obtained at 24 hours after treatment. Blood samples from five patients were drawn more frequently during the first 2 hours of a subsequent treatment course. The preadministration plasma levels of plasminogen activator enzyme activity, tissue-type plasminogen activator antigen, and plasminogen activator inhibitor were 1.433 ± 0.411 IU/mL (mean ± SE), 1.546 ± 1.381 ng/mL, and 2.162 ± 1.284 AU/mL, respectively. Plasminogen activator enzyme activity and levels of tissue-type plasminogen activator antigen increased beginning at 20-30 minutes after TNF Statistical Methods initiation, while levels of plasminogen activator inhibitor remained stable for 2 Two kinetic series of data were available for analysis: the 24-hour series and the 2-hour series. hours. At 1 hour, plasminogen activator Not all patients had specimens available for each activity and levels of tissue-type plastime. Repeat series were excluded from the minogen activator antigen showed 7.3statistical analysis because there were too few of them, but they were used to corroborate the and 30.8-fold increases, respectively, findings noted. In cases where two or more over the baseline concentrations. At 6 series were obtained, data from the first available hours, the plasminogen activator inhibiseries were used for statistical analysis. All data tor peak activity was elevated 38.6-fold were from day 1 of a treatment week. Three of over baseline. Levels of tissue-type five 2-hour series had concomitant blood cell plasminogen activator antigen remained counts. All statistical analyses were based on pairwise elevated for 4 hours and gradually Plasminogen activator differences. We either compared a value after declined. infusion with its pretreatment level or compared enzyme activity dropped sharply at 2 two consecutive observations by Wilcoxon hours to just above baseline levels; at signed rank test. Differential dose-response effects of TNF for three or more groups were the same time, concentrations of plasanalyzed by applying the Jonckheere-Terpstra minogen activator inhibitor rose (Fig. test (44) to differences between values and 1, A and B). baseline (time 0). The Jonckheere-Terpstra test is The assay for plasminogen activator more sensitive to trend with increasing dose inhibitor quantitates the net plaseffect than an overall test of differences. Dose minogen activator inhibitor activity in effects of two groups were tested by a permutation test of 6-hour response profiles and by a plasma. Therefore, a rise in levels of Wilcoxon two-sample test applied to paired plasminogen activator inhibitor with a differences and to the medians at each time. concurrent, rapid fall in plasminogen Temporal associations between end points were examined by computing Spearman correlation activator enzyme activity indicated that plasminogen activator inhibitor in coefficients among differences. plasma had neutralized the catalytic activity and exceeded the levels of free Results plasminogen activator enzyme. This pattern was observed in most patients. TNF-Associated Changes in Samples were obtained from seven Circulating Plasminogen Activator patients 24 hours after TNF infusion. and Plasminogen Activator Both plasminogen activator activity and Inhibitor tissue-type plasminogen activator antiFourteen separate sequential observa- gen returned to basal levels in all tions were available from 14 patients patients, but plasminogen activator in(SDS)-PAGE vertical slab electrophoresis procedure of Laemmli (42) was followed. We used 3% stacking gels and 10% separating gels of 1-mm thickness and 50-mm length. Thirty microliters of each sample were mixed with an equal amount of sample buffer and electrophoresed under nonreducing conditions at 15-mA constant current. Room temperature was maintained at 22 °C ± 1 °C. Molecular mass markers of 14 400 to 97400 daltons (catalog No. 161-0304; Bio-Rad, Richmond, Calif.) were included for the determinations of the apparent molecular weights. The marker lane was cut away after electrophoresis and stained with Coomassie Blue G 250 to visualize the proteins. The remaining gel was rinsed with 2.5% Triton X-100 for 1 hour, followed by two rinses of 10 minutes each with distilled water. It was then drained and layered onto a casein-agar zymography gel that contained 2% casein, 1.2% agar, and 18 p.L of plasminogen per milliliter of 0.1 M Tris-HCl buffer (pH 8.1) (43). After the layering step, we incubated the remaining gel at 37 °C and periodically examined it under dark-field illumination for zones of lysis. The results were photographed.

Journal of the National Cancer Institute

Downloaded from http://jnci.oxfordjournals.org/ at OCLC on July 26, 2015

18-24 hours after initiation of TNF infusion on the 1st day of a treatment week. (Hereafter, this protocol is called the 24-hour series.) Five separate series of specimens were obtained during the 2-hour TNF infusion—at 0, 1, 2, 5, 10, 15, 20, 30, 60, 80, and 120 minutes. (Hereafter, this protocol is called the 2-hour series.) Sodium citrate was used as an anticoagulant in the blood samples, which were transported to the laboratory within an hour of venipuncture. Two of the 2-hour series were not immediately processed, but the effect of delayed processing appeared to be negligible. The samples were centrifuged at 2000g for 20 minutes at 4 °C, stored at -70 "C in 1-mL aliquots, and thawed for analyses. Plasminogen activator was assayed with a chromogenic substrate method for total enzymatic activity and by an immunoassay method for tissue plasminogen activator antigen. The enzymatic assay utilized the tripeptide H-D-ValLeu-Lys-p-nitroaniline-2HCl (catalog No. 5277; Helena Laboratories, Beaumont, Tex.) as the substrate for plasmin generated from plasminogen by plasminogen activator action, and amidolytic activity was measured by the release of p-nitroaniline at 405 nm (35,36). The enzymatic activity for plasminogen activator in plasma was expressed as international units (IU) per milliliter, in comparison with purified tissue-type plasminogen activator of 500000 IU of activity per milligram of protein as the standard. Samples were thawed rapidly at 37 °C just prior to assay for plasminogen activator and treated with acid by addition of 100 JJLL of 0.1 M sodium acetate buffer per 100 u,L of plasma and incubation at 37 °C for 15 minutes.

obtained during two cycles of TNF administration scheduled at least a week apart; one series was obtained on 25. 25. day 5 of a treatment week). Comparison of these values revealed that all 320. 20. three patients had similar basal levels. i.15. 15, In all three patients, however, the a. magnitude of the levels of plasminogen = 10.| activator activity and tissue-type plasminogen activator antigen was consisto ently lower in the second cycle (data not shown). Although sample sizes were small 0 i 4 9 12 15 1'B 21 2*4 i 204fl 60 80 iAo 120 (six to 12), some pronounced patterns Hours After Start of TNF Infusion Minutes After Start of TNF Infusion 100, 100, of correlation emerged. There was a strong negative correlation (Spearman 80. B0. correlation r = -.8) between increased levels of tissue-type plasminogen ac60. tivator antigen from time 0 to 1 hour after TNF treatment and increased a. 40, 40. levels of inhibitor from 1 to 2 hours after treatment. A scatter plot based on I 20, 20. these parameters demonstrated two disa tinct groups of patients (Fig. 2). Four 0 patients had an appreciable increase in •MM plasminogen activator inhibitor in this 0 3 6 9 12 15 18 2i 24 6 20 40 60 80 100 120 interval and very low responses of Hours After Start of TNF Infusion Minutes After Start of TNF Infusion 120, 120, tissue-type plasminogen activator antigen. All four of these patients received 100, high doses of TNF (3=200 |i.g/m2). Samples from three of these four 3 80, patients were measured at 24 hours and still showed elevations of plasminogen £ 60, activator inhibitor. Conversely, the suba. c 40, stantial elevations in tissue-type plasminogen activator antigen at low doses did not lead to appreciable levels of £ 20. plasminogen activator inhibitor at 1-2 hours. 0 3 6 i 12 15 18 21 2« 0 20 40 60 80 100 120 Small sample size limits the ability Hours After Start of TNF Infusion Minutes After Start of TNF Infusion to accurately analyze the results by Fig. 1. A) Mean changes from baseline to 1, 2, 4, 6, and 24 hours after TNF initiation for dose of TNF. Statistical analysis of plasminogen activator enzyme activity (PA), tissue-type plasminogen activator antigen (tPA), and changes of differences by dose over plasminogen activator inhibitor (PAI). Plotted points are the means of up to 12 patients, depending on four levels failed to show statistically the number of possible data pairs. B) Minute-to-minute analysis during TNF infusion of plasminogen activator enzyme activity, tissue-type plasminogen activator antigen, and plasminogen activator significant results. However, when painhibitor in five patients. Mean changes from baseline to 1, 2, 5, 10, 15, 20, 30, 60, 80, and 120 tients were divided into low-dose minutes after TNF initiation for plasminogen activator enzyme activity, tissue-type plasminogen (

Plasminogen activator and its inhibitor in cancer patients treated with tumor necrosis factor.

We noted the presence of plasma fibrin degradation products in patients treated with recombinant human tumor necrosis factor (TNF) in a phase I trial...
4MB Sizes 0 Downloads 0 Views