Correlation of the in vivo anticoagulant, antithrombotic, and antimetastatic efficacy of warfarin in the rat

THROMBOSIS RESEARCH 50; 163-174, 1988 0049-3848188 $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

CORRELATION OF THE -IN VIVO ANTICOAGULANT, ANTITHROMBOTIC, ANTIMETASTATIC EFFICACY OF WARFARIN IN THE RAT

AND

Gerald F. Smith, Blake L. Neubauer, Jacqueline L. Sundboom, Kevin L. Best, Robin L. Goode, Lee R. Tanzer, Ronald L. Merriman, J. D. Frank, and Roy G. Herrmann Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285 U.S.A.

(Received 2.2.1987; Accepted in revised form 21.1.1988 by Editor J. Hawiger) ABSTRACT Fibrin formation has been hypothesized to be an element of the metastatic process in cancer, and pharmacological interference with such fibrin formation has been proposed as a means of antimetastatic therapy. We have tested this hypothesis through an -~ in vivo study of warfarin in two independent rat disease models - a model of chemical-injury-induced arterial thrombosis, and a model of spontaneous metastasis. We found 0.50 mg/kg-day warfarin to be uniformly lethal after two weeks treatment. The chronic dose of 0.25 mg/kg-day was non-toxic and produced effective anticoagulation and marked antithrombotic and antimetastatic activity. The 0.125 mg/kg-day dose produced a reduction in factor IIc (50%) and factor VIIC (70%), and resulted in statistically significant antithrombotic and antimetastatic activity. The 0.0625 mg/kg-day dose failed to reduce the vitamin K-dependent clotting factors, and or antimetastatic effects. failed to produce any antithrombotic (very similar dose-response effects) The substantial correlation antimetastatic among the anticoagulant, antithrombotic and efficacies of warfarin in the rat suggests that anticoagulation provides the pharmacological mechanism underlying both the antiThe poor therapeutic thrombotic and the antimetastatic effects. index we observed in the rat may be the attribute which limits the efficacy of warfarin in the treatment of human cancer.

INTRODUCTION For many years clinicians have noted the association between thrombosis The central role of blood coagulation in the inflammatory and cancer (l-3). response and in healing processes (4,5) provides a general biological basis

Key Words:

Thrombosis,

warfarin,

metastasis,

163

anticoagulation,

cancer

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WARFARIN AS AN ANTIMETASTATIC

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for the concept that blood coagulation is important in cancer processes (6,7). A number of laboratories are attempting to elucidate the mechanisms involved in the blood coagulation-cancer interrelationships (8-lo), and to consider these in the development of anticancer pharmaceutical agents (11,12). Sufficient evidence exists in the clinical and experimental literature to warrant the hypothesis that fibrin formation is a requirement for tumor cells to succeed in resisting the host defense mechanisms or to successfully perform certain functions (7-18) which contribute to metastasis (19-23). Therefore, the pharmacological inhibition of fibrin formation could produce anticancer efficacy, especially that of antimetastatic activity. There have been successful demonstrations of the in vivo anticancer activity of oral anticoagulants (coumarin derivatives which antagonize the biosynthesis of vitamin K-dependent clotting factors) in animal models (24-27) and in human therapy (12,28-30). Warfarin has proved to be beneficial in the treatment of small cell lung carcinoma (28) and advanced breast cancer (30), but its efficacy and its value in other cancers remains unclear (28). This report describes our initial studies in two distinct rat disease models which allow us to compare the anticoagulant, antithrombotic and antimetastatic effects of warfarin. MATERIALS

AND METHODS

The rats were housed in raised wire cages and fed ad libitum Animals. powdered Purina No. 5001 chow, and given water ad libitum. Lighting ___ was controlled (on: 6:00 a.m.; off: 8:00 p.m.). Animals ~ used in the antimetastasis model were male Lobund Wistar rats maintained as a closed colony at Harlan Industries (Cumberland, IN) from breeding stock donated by Dr. Morris Pollard (University of Notre Dame). Animals for the FeC13 arterial thrombosis model were male Sprague-Dawley rats from Charles River (Wilmington, MA). Sodium warfarin (Abbott Laboratories, lot 20-161Warfarin Administration. AF) was administered in daily oral doses by blending appropriate amounts into the powdered chow. The warfarin doses tested and the treatment durations are given with the results of the various experiments. Blood Coagulation Techniques. All blood samples were obtained from the animals by cardiac puncture (plastic syringes), under Metophane (PittmanMoore, Inc.) vapor anesthesia, and immediately treated with 1 volume 3.8% Blood coagulation assays trisodium citrate dihydrate per nine volumes blood. We have were performed immediately using whole blood as the substrate. found, in agreement with the literature (31), that whole blood coagulation assays provide results comparable to those obtained using plasma samples (we found such comparability in dose-response studies with heparin, warfarin and Whole blood coagulation assays were performed experimental anticoagulants). using a Fibrometer (BBL). For whole blood prothrombin time (WBPT) assay we mixed 0.1 ml blood, 0.1 ml NaCl (0.15 M), and 0.1 ml thromboplastin C reagent (Dade). For whole blood activated partial thromboplastin time (WBAPTT) assay (0.02M). we mixed 0.1 ml blood, 0.1 ml Actin reagent (Dade), and 0.1 ml CaCl For whole blood recalcification time (WBCT) assay we mixed 0.1 ml l?lood, 0.1 For whole blood thrombin time ml NaCl (0.15 M), and 0.1 ml CaCl (0.02 M). assay we mixed 0.1 ml blood, 0.1 m 1 NaCl (0.15 M), and 0.1 ml bovine thrombin Factor IIc and factor VIIc were assayed in (Parke Davis, 7 N.I.H. units/ml). test sample to 0.1 ml blood samples by adding 0.1 ml of saline-diluted factor-free human plasma (Dade) and then measuring the WBPT by adding 0.1 ml

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WARFARIN AS AN ANTIMETASTATIC

thromboplastin C reagent. Standard data curves were obtained using salinediluted whole blood from control animals and by electing the l/10 salinediluted blood to be 100% of normal. PA111 Prostatic Adenocarcinoma Model in the Rat. This model involves an autochthonous tumor grown in cell culture and implanted into the tail of male Lobund Wistar rats. An adenocarcinoma grows at the site of implantation and subsequently metastatic lesions develop in the lymph nodes and in the lungs. The details of the procedure have been recently published J32-35). The animals are maintained for 29-30 days after implantation of 10 tumor cells, then sacrificed for measurement of primary tumor burden (tail segment weight) and metastatic tumor burden. The metastatic spread into the lungs is measured by microscopic determination of lung colony number (32-35). Test agents are evaluated in this model by administering the experimental drugs at desired times relative to the implantation date and by measuring the spread of the tumor in the animals. Chronic warfarin treatment was begun either on the day of implantation or seven days prior to implantation. Blood samples were obtained for coagulation assays on day 29 or 30. FeCl, Arterial Thrombosis Model in the Rat. The experimental details are J essentially those used in a well-defined technique where occlusive carotid arterial thrombosis (subsequent to arterial injury) is accurately detected by monitoring the rat carotid arterial temperature with an thermocouple connected to a Tele-Thermometer (36,37). Occlusive thrombosis occurs with an abrupt 1.5-2.5OC fall in temperature in the rat carotid artery (36,37). We modified this technique by substituting a chemical injury to the exposed carotid artery for injury from local electric current. Male Sprague-Dawley rats, 350-400 g, were anesthetized with pentobarbital, 40 mg/kg i.p. The carotid arteries were exposed surgically, freed of connective tissue, and the test artery laid across a thermistor (Yellow Springs Instrument Co. Model 427). Temperature of the artery was continuously monitored by connection to a Tele-Thermometer (Yellow Springs Model 435D), using a strip chart recorder (Hewlett-Packard Model 680). Chemical2 injury . to the exposed artery was accomplished by applying a segment (3mm ) of Whatman 111 filter paper on top of the exposed artery and adding onto the paper 10 ~1 of 5% FeC13 (in saline). The time elapsed from application of the chemical solution until occlusive arterial thrombosis is indicated by an abrupt temperature fall (1.5-2.5OC) on the strip chart and this time is termed the "occlusion" time (36,37). The test is performed twice in each animal, first in the left carotid artery then in the right carotid artery. The control (no treatment) value of the occlusion time in this model has been 21 f 2 minutes (150 control animals). Warfarin was administered daily beginning at least nine days prior to the The warfarin anticoagulation effects were measured in the thrombosis test. rats immediately after the second occlusion time measurement. Statistical Methods. comparison procedure

The (38).

data

were

analyzed

using

Dunnett's

multiple

RESULTS The Efficacy of Warfarin as an Anticoagulant in the Rat Preliminary experiments determined the proper anticoagulant dose range to compare the antithrombotic and antimetastatic efficacy of warfarin in The LDIOO dose of warfarin in these rats is 0.5 the male rat (Table 1). mg/kg-day - about one half of a group died by day 10, the remainder died by In marked contrast, the day 14-21 (all animals showed hemorrhagic signs). warfarin dose of 0.25 mg/kg-day is safe, with the pharmacological endpoints

Lobund Wistar (28 Days)

Sprague-Dawley (5 Days)

B.

c.

(5) (5) (5) (5)

(4) (4) (4) (4)

(4) (4) (4) (4) (4)

(n)

0.250 0.125 0.0625 0

0.250 0.125 0.0625 0

0.500 (toxic) 0.250 0.125 0.0625 0

Warfarin Dose (mg/kg-day)

_-

"35 19 18 19

+ 2 + 1 i: 1 ? 1

WBPT(sec)

24 + 2 22 + 1 "44 + 3 "26 + 2 18 + 1 22 -L 1

21 ? 1 20 ? 1 "31 2 2 22 r 2 18 + 1 19 + 1

'745 2 25 + 22 + 22 +

4 1 1 1

_-

n150 f 19 "75-+ 5 65? 8 57+ 5

61% 60?

';7+ 1 ;'; 56 f 12 115 f 12 103 ? 21

6 5

137 + 13 >'i81* 5

"141 + 11 712 5 68+ 6 62+ 5

__

::- 7 -'-30 101 97

+ 3 + 25 + 25 t 19

Factor VIIc(%) ____-__

"127 + 11 782 6 73+ 6 70+- 6

"136 ? 12 "99+ 8 73+ 5 68+ 6

__

WBCT(sec) Day 14 Day 28

Factor ___ IIc(%)

28 f 3 26 + 2 25 + 2

~'42 * 3

27 + 3 22 + 2

"48 + 3 "34 + 3

WBCT(sec)

"53 + -- 4 :';28_+ 2

"35 _-_+ 3 "25 + 1

WBAPTT(sec)

23 f 2 18 r 1 20 + 1

;';44+ 2

23 ? 2 20 ? 1 20 t 1

"38 k 3

in the Rat

WBAPTT(sec) Day 14 Day 28

Efficacy

WBPT(sec) Day 14 Day 28

Anticoagulant

astatistically significant standard deviation.

difference

from untreated

control group (~~0.05); data show mean values ?

Tin studies A and B, the animals survived heart puncture on Day 14 and were further treated through day 28 All coagulation assays were performed in when blood samples were obtained again from the same animals. duplicate.

Lobund Wistar (28 Days)

A.

Study-f

Warfarin

'Table 1

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WARFARIN AS AN ANTIMETASTATIC

of 1% to 2x prolongation of the prothrombin time maintained steadily throughNo hemorrhagic signs could be detected at this out at least a 35 day period. dose. Thus, the therapeutic index of warfarin is very narrow in the rat. The 0.125 mg/kg-day warfarin dose caused no changes in the conventional blood coagulation assays. However, measurement of factors IIc and VIIc revealed that this warfarin dose depressed these two vitamin K dependent proteins by 50% and 70x, respectively - even though the prothrombin time was not altered (Table 1 - Study C). The warfarin dose of 0.0625 mg/kg-day did not influence the factor IIc and factor VIIc levels. The Antithrombotic Efficacy of Warfarin in the Rat Male Sprague-Dawley rats were chronically treated with warfarin for subsequent testing in the FeC13 thrombosis model. Pairs of animals from the We invarious dose groups were taken on days 9-11 for thrombosis testing. cluded a dose group at 0.50 mg/kg-day (the LDIO dose) since there would be sufficient survivors at 9-11 days for testing. % lood coagulation assay data were obtained from cardiac puncture blood samples taken immediately following We independentthe occlusion time measurements (See Materials and Methods). ly determined that the thrombosis test procedure does not alter the coagulation assay data obtained from either untreated or warfarin-treated animals. Table 2 Antithrombotic

Warfarin Dose (9-11 Days) (mg/kg-day) (n)

and Anticoagulant Efficacy of Warfarin Carotid Arterial Thrombosis Model

WBPT(sec)

WBAPTT(sec)

0.500 0.250 0.125

(4) (10) (4)

;?45 2 2 "32 2 6 19 + 1

"56 2 3 "39 & 3 lost

0.0625 0

(4) (10)

18 + 1 20 + 1

lost 24 ? 1

WBCT(sec) "200 + 3 "106 + 14 702 5 652 602

3 4

in the Rat

Arterial Occlusion Time(min) ">60 ir;46 k 9 7';32 + 7 26 + 6 21 f 2

*See Table 1. Table 2 presents the antithrombotic and anticoagulant efficacy of The anticoagulant effects arterial thrombosis model. warfarin in the FeCl shown in Table 2 (9111 days of treatment) are consistent with the results described in Table 1. The high dose of 0.50 mg/kg-day completely prevented arterial thrombosis (throughout the maximal observation time of 60 min). The dose of 0.25 mg/kg-day warfarin (Table 2) showed a marked delay in the occlusion time (until 46 min), presumably because of the significant antiThe warfarin dose of 0.125 mg/kg-day coagulation achieved in these rats. caused a significant inhibition of arterial thrombosis in this model even though no anticoagulation effects were measured by the conventional coagulation assays. However, the blood concentrations of factor IIc and VIIc were This reduced to 56% of normal and 30% of normal (see Table 1 - Study C). suggests that the depressed levels of factor IIc and VIIc are related to the The lowest dose of warfarin (0.0625 antithrombotic efficacy of warfarin. mg/kg-day) produced no anticoagulant effects, no reduction of factor IIc or These results suggest a effects. VIIc (Table l), and no antithrombotic and the antithrombotic dosestrong corelation between the anticoagulant response effects of warfarin in the rat.

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+I

+I

m-Fcp;Dlhl 2:

t

+t

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WARFARIN AS AN ANTIMETASTATIC

169

The Antimetastatic Efficacy of Warfarin in the Rat Warfarin was evaluated for antimetastatic efficacy in the PA111 prostatic adenocarcinoma model by using the same (vide supra) warfarin dose range. In the two separate experiments shown in Table 3 warfarin administration was begun either on the day of (Table 3, A), or six days prior to (Table 3, B), tumor cell inoculation. In both experiments, cardiac puncture blood samples for coagulation studies were obtained on post-inoculation day 30, immediately before the animals were sacrificed and evaluated for primary and metastatic lesions (see Methods). The primary tumor burden (measured by tail weight) in the animals was not different in the warfarin treated animals as compared to untreated animals (data not shown). In contrast, warfarin did reduce pulmonary metastasis in a dose-dependent fashion (Table 3). The only warfarin dose which failed to produced a statistically significant prevention of lung metastasis was the dose of 0.0625 mg/kg-day - the dose which also fails to produce measurable anticoagulation effects in the rat (even when assayed for factor IIc and factor VIIc effects). The antimetastatic effect of warfarin was the same whether warfarin treatment was begun on the day of tumor cell inoculation or begun one week prior to inoculation. In either circumstance, the 0.125 mg/kg-day warfarin dose caused statistically significant reduction in the number of lung tumor foci. The fully anticoagulating warfarin dose of 0.25 mg/kg-day inhibited lung tumor foci development by 51% - 72% of that found in the untreated PAIII- bearing animals. All animals treated with the This 0.50 mg/kg-day dose died prior to the 30 day test termination date. dose group was included to empirically establish the therapeutic index of warfarin in the cancer model and to preclude the possibility that the disease might alter the drug toxicity. DISCUSSION The findings presented in this report suggest a substantial relationship among the anticoagulant efficacy, the antithrombotic efficacy, and the antimetastatic efficacy of warfarin in the rat. In a well-defined rat model of arterial thrombosis (36,37), warfarin inhibited arterial thrombosis such that the dose-response effects were related to the anticoagulant effects. Likewise, the same doses of warfarin inhibited the spread of tumor in a well-defined rat model of spontaneous metastasis (30,32,34) such that the The largest dose-response effects were related to the anticoagulant effects. non-toxic warfarin dose tested (0.25 mg/kg-day) produced fully effective anticoagulation and markedly inhibited both arterial thrombosis and spontaneous lung metastasis. These corresponding -in vivo effects suggest that warfarin's inhibition of the blood coagulation system provides a mechanism for the antithrombotic and antimetastatic activity in two distinct rat models. Inhibition of the blood coagulation system has the functional conseSuch inhibition would be expected quence of inhibiting fibrin elaboration. to reduce thrombosis in the fibrin-dependent thrombosis model (36,37) which we utilized. However, the PA111 metastasis model has not been so characterized to be dependent upon -in situ fibrin formation, and an anticoagulant Our effect would not be readily expected to provide antimetastatic effects. study was based upon the idea that PA111 model tumor metastasis could be dependent upon fibrin elaboration, according to the modern hypothesis which proposes that fibrin formation is necessary for the tumor cell's ability to Fibrin formation may simply provide a means for metastasize (3,6,8,9,19-23). the tumor cell to evade the host's defense mechanisms (25,25) and/or may

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provide molecular requirements for certain functions of the tumor cell [e.g., adherance (15,16), angiogenesis (13,14)] necessary to successfully invade and spread. Fibrin is an active principle in healing processes (14), therein attracting fibroblasts (39), leukocytes (13), and macrophages (13), promoting the formation of granulation tissue (39), and promoting angiogenesis (13,14). Fibrin has been shown to induce changes in cultured endothelial cells which would lead to vessel permeability and cell organization consistent with angiogenic mechanisms (40-42). Tumor cells can elicit fibrin formation either intravascularly (24) or in the interstitial (7,8) compartments, by their ability to express tissue thromboplastin (3,6,22) or other procoagulants (9,23). Therefore, this modern hypothesis would provide the explanation of the observed antimetastic effects of warfarin in the PA111 model as a consequence of inhibiting the ability of the tumor cells to elicit required fibrin formation. The fact that we found corresponding dose-response effects for the anticoagulant, antithrombotic, and antimetastatic effects is consistent with, though not proof of, such an explanation for warfarin's antimetastatic efficacy. We observed no effects of warfarin upon the primary tumor growth in the spontaneous metastasis model. We found the same antimetastatic effect of warfarin whether we pre-anticoagulated the animals one week prior to tumor cell inoculation (Table 3, B), or whether we instituted warfarin therapy on the day of tumor implant (Table 3, A). The latter treatment involves a three day period without maximal anticoagulation following implantation (the time required for warfarin to exert its anticoagulant effect is about three days), anticoagulation before while the former treatment provides maximal inoculation. Therefore, the "take" of the implanted tumor cells, like the growth of the primary tumor following implantation, was not affected by The beneeither previous anticoagulation or postimplant anticoagulation. ficial effect of warfarin in this metastasis model appears to be confined to inhibition of tumor spread. Our study, while demonstrating the antimetastatic effects of warfarin, also points to pharmacological considerations which may explain the efficacy limitations which have been observed with the use of warfarin in human cancer (28-30). The therapeutic index of chronic warfarin treatment in the rat is index observed may apply to other very narrow. The narrow therapeutic Indeed, the anticoagulant potency of warfarin species, including the human. is not markedly different among the rat, mouse, guinea pig and dog (43-45), and the maintenance dose in man is about 0.1 mg/kg-day (46). Warfarin could prove to be more useful in the treatment of human cancer if treatment were the dose-anticoagulation balance, control of rigorous undertaken with including control of the nutritional and drug perturbations of the vitamin K We suggest that other agents which inhibit fibrin formastatus (43,44,46). tion should be considered as candidates for new antimetastatic pharmaceutical agents. REFERENCES Clinique medicale Phlegmasia alba dolens. New Sydenham Sot., 2, 94, 1865. London.

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