Pharmacological evaluation of combination therapy of P388 leukemia with cyclophosphamide and pyrimidinones

Int. J. lmmunopharmac., Vol. 9, No. 1, pp. 3 1 - 3 9 , 1987. Printed in Great Britain.

0192 0561/87 $3.00+ .00 International Society for lmmunopharmacology.

P H A R M A C O L O G I C A L E V A L U A T I O N OF C O M B I N A T I O N T H E R A P Y OF P388 L E U K E M I A W I T H C Y C L O P H O S P H A M I D E AND PYRIMIDINONES L. H. Ll, T. L. WALLACE, R. D. HAMILTON and T. F. DEKONING Cancer and Viral Diseases Research, The Upjohn Company, Kalamazoo, MI 49001, U.S.A. (Received 1 May 1986 and in final form 1 July 1986)

Abstract - - The administration of pyrimidinone prior to tumor inoculation did not increase the host

resistance to tumor invasion. In addition, the administration of pyrimidinone prior to cyclophosphamide (CY) was no better than the treatment with CY alone. The best therapeutic effect was observed when the pyrimidinone was given 1 day after CY administration. A statistically significant synergism (mostly P < 0.01) with CY was obtained when the pyrimidinone was given every 4 days thereafter for 1 - 7 intraperitoneal (i.p.) injections, A significant synergistic antitumor effect was also observed when the pyrimidinone was given at 4 day, 7 day, 10 day, or 14 day intervals for a maximum of 7, 4, 3 and 2 injections, respectively. For some pyrimidinones, such as ABPP, multiple injections appeared to yield better therapeutic results. This phenomenon may be in part related to the magnitude and duration of the stimulation of natural killer (NK) cell activity after mice received a single or multiple i.p. injections of ABPP. When given i.p., the pyrimidinone level in peritoneal exudates was approximately twice that found in serum. CY showed no apparent effect on the absorption and clearance of pyrimidinone. The combination therapy was also statistically synergistic (P < 0.01) when ABPP was given orally. The significance and advantage of these findings is discussed in terms of their clinical implications.

We have demonstrated a marked synergistic antitumor effect against P388 leukemia (Li, Johnson, Moeller & Wallace, 1984) L1210 leukemia and M5076 tumor (Li, Wallace, Wierenga & DeKoning, 1985) when the pyrimidinone was used in conjunction with CY even though pyrimidinones by themselves showed little or no activity against these tumors. The synergistic effect appeared to be related to, at least in part, the initial t u m o r burden reduction by CY and the marked enhancement of both natural killer cell and macrophage activities by the pyrimidinone (Li, Wallace, Richard & Tracey, 1985). The aim of this study was to evaluate the pharmacological characteristics of selected pyrimidinones in an attempt to optimize the conditions for the m a x i m u m therapeutical effect against P388 leukemia when used in combination with CY.

Pyrimidinones, including A B P P (2-amino-5-bromo6-phenyl-4(3H)-pyrimidinone), A B M F P P (2-amino-5bromo-6(3- fluorophenyl)-4(3 H)-pyrimidinone) and A I M F P P (2-amino-5-iodo-6(3-fluorophenyl)-4(3H)pyrimidinone) were made at The U p j o h n C o m p a n y by published procedures (Wierenga, Skulnick, Stringfellow, Weed, Renis & Eidson, 1980) and the structures are shown in Fig. 1. These compounds were prepared in fine suspension with Sterile Vehicle 100 (SV-100), prepared for laboratory use at The U p j o h n C o m p a n y , Kalamazoo, MI, U . S . A . , prior to injection into the animal. SV-100 is composed o f 5 mg carboxymethylcellulose, 4 mg polysorbate 80, 9 mg sodium chloride, and 9 mg of benzyl alcohol in 100 ml vehicle.

A n i m a l s and tumors Male BDF~ (C57BL/6 female × D B A / 2 male) and male CDF~ ( B A L B / c female × D B A / 2 male) mice were supplied by the National Cancer Institute Frederick Cancer Research Facility, Frederick, MD, U.S.A. These mice were generally 5 - 6 weeks of age and weighed 1 8 - 2 2 g. Groups of eight to ten mice

EXPERIMENTAL PROCEDURES

Agents and vehicles CY (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in sterile saline solution. 31

32

L. H . LI

Br•N•[

INH2 NH

O ABPP

FN B ~ r iI N~'1.'Y""NH2 NH

0

ABMFPP

F - J ~ N ~ . . - NH2 0

AIMFPP

Fig. 1. Structures of pyrimidinones. were housed in metal .suspended cages and were given pelleted food and water ad libitum. P388 leukemia was maintained by continuous i.p. passage in syngeneic DBA/2 female mice.

Experimental protocol P388 leukemia cells (106 cells/mouse) were inoculated i.p. on day 0. One day later the animal received a single i.p. injection of CY. The dosage, route and/or schedule of pyrimidinone administration, which was the main subject of this investigation, will be described in detail in the "Results" section. The therapeutic response was measured as median survival time (days) of eight to ten mice per group according to the procedure of Schabel, Griswold, Laster, Corbett, & Lloyd (1977), in which the median life span was determined with the dying animals only. The long-term survivors (30 days or longer) were recorded separately. Percent increase of life span (ILS) was calculated as median survival time (days)of treated group_ 1"~× 100. median survival time (days) of control group According to the National Cancer Institute (U.S.A.) (Geran, Greenberg, MacDonald, Schumaker & Abbott, 1972), the criterion for significant therapeutic effect is >= 27o7o for P388 leukemia (i.p.). Acute drug toxicity was estimated by the early deaths of animals and the change in animal body weight which was measured on days 1, 5, and 9 for BDF~ mice bearing P388 leukemia.

et al. Statistical analysis Drewinko, Loo, Brown, Gottlieb & Freireich (1976) defined an additive therapeutic effect as one for which the fraction (treatment divided by control) of surviving cells under joint therapy equals the product of the fractions of surviving cells under the individual therapies. Schabel et al. (1977) showed that median mouse life span was linearly related to the log of the number of inoculated cells if survivors were not included in the calculation of the median. Consequently, Drewinko's definition of additivity in terms of the fraction of surviving cells is equivalent to defining an additive effect as one for which the median of the log (life span) under joint therapy equals the sum of the effects of the individual therapies on the log (life span). A synergistic effect is any effect exceeding this expected additive effect. These latter definitions of additive and synergistic effects require that survivors and mice with spuriously low spans be appropriately removed from the analysis. Median and mean life spans were calculated after removing survivors (>30 days for P388 leukemic mice) and spuriously low life spans. Working with the log of the life span, spuriously low life spans for the control group were removed using Dixon's gap test (Johnson & Leone, 1964). Any life span of a treated group which was <3 S.D. below the control mean was also excluded. Since the distribution of life spans is roughly symmetrical after survivors are excluded, the medians can be replaced by the means in the preceding definitions. Consequently, a general linear model (Draper & Smith, 1981) could be used to set up the contrast that the sum of the expected log (life spans) for the control and joint therapy groups equals the sum of the expected log (life spans) for the individual treatment groups. A positive and statistically significant contrast implies a synergistic effect. Statistical significance was based on the F-test for interaction (Snedecor & Cochran, 1967), a robust and powerful test (Pearson & Please, 1975) for location differences. Determination o f pyrimidinone levels in biological fluids Sera and/or peritoneal exudates were collected and pooled at 1, 2, 4, 6, 8, 12, 24, and/or 72 h after the mice received a single i.p. injection of pyrimidinone. The peritoneal cavity was washed once with 2.5 ml of Sterile Vehicle 100 or sterile water and pooled from two to five mice. The pyrimidinone levels in these biological fluids were

Pharmacological Evaluation of Pyrimidinones determined by high-performance liquid chromatography according to the procedure of Wynalda & Fitzpatrick (1980)

33 I

CY CY + ABMFPP * p<0.01

~5o

Assay for cytotoxicity of natural killer (NK) cells The procedure has been reported previously (Li et al., 1985). Essentially, effector cells prepared from peritoneal exudates were adjusted to 9 x 105 cells/ ml, serial three or four-fold dilutions were made with R P M I 1640 medium, then 0.1 ml of each cell suspension was added to triplicate wells of a 96-well V-bottomed microtiter plate. The labeled YAC-1 target cells were adjusted to 5 x 104 cells/ml and 0.1 ml of the cell suspension was added to the appropriate wells. Thus, the final effector cell to target cells ratios would be 18, 6, 2 and 0.67. The plates were covered and centrifuged at 200 x g for 6 min and then incubated at 37°C for 3 - 4 h. After incubation, the plates were centrifuged again and 0.15 ml of the supernatant was removed from each well, transferred to glass tubes and counted in a Packard g a m m a scintillation counter. The specific cytotoxicity of NK cells was calculated according to the following equation %

. . o f c y t OtOXlClty

test counts/min = total counts/rain

-- spontaneous " -- spontaneous

release counts/min release counts/min

X

*~

100

E

i

iso 2

4

6

8

Day

Fig. 2. Determination of the optimal gap between CY and pyrimidinone administration. P388 leukemia cells (106/ mouse) were inoculated i.p. on Day 0, and CY was injected i.p. on day I. Sterile Vehicle 100 or pyrimidinone was injected i.p. on day 1, 2, 4, or 8 and every 4 days thereafter for a maximum of seven injections. The median death of vehicle-treated tumor-bearing mice (control) was 10.5 days.

100

where test c o u n t s / m i n , spontaneous release c o u n t s / min and total counts/rain represent the radioactivity in 0.15 ml aliquots of the supernatant from the effector and target cell mixture, the supernatant of target cells only and resuspension of target cells, respectively. Each microtiter plate contained at least one triplicate set of wells for the determination of total radioactivity and spontaneous release of radioactivity.

ABMFPP ( 1 2 5 m g / k g , i.p.) • - CTX • + CTX

E E .E E

RESULTS

20

15

== •~ 10

Determination of the optimal gap between CY and pyrirnidinone adm&istration Mice received a single i.p. injection of CY (100 m g / k g ) on day 1 and A B M F P P (125 m g / k g / injection) starting on day 1, 2, 4, or 8 and every 4 days thereafter for a m a x i m u m of seven injections. The simultaneous addition o f both agents did not produce the best therapeutic effect (Fig. 2). As a matter of fact, A B M F P P given 1 day after CY administration appeared to be the best, although a statistically significant synergism (P<0.01) was observed whether the administration of A B M F P P was started on day 1, 2 or 4. The synergistic effect was diminished as the gap between CY and pyrimidinone administration increased and it was no

Time (hr)

Fig. 3. Determination of serum levels of ABMFPP in CDF, mice when administered alone or in combination with CY. A single dose of ABMFPP was given immediately after a single i.p. injection of CY (100 mg/kg). The ABMFPP levels were determined by high-performance liquid chromatography according to the procedure of Wynalda & Fitzpatrick (1980).

L. H. LJ et at.

34

Table 1. influence of schedule of pyrimidinones treatment No. of injections t

Treatment* SV-100 ABMFPP (125 mg/kg/injection)

ILS(%) SV-100

7 7 2 9 7 2 9

AIMFPP (100 mg/kg/injection)

0 10 0 0 10 5 25

CY* 110 150 105 150 140 110 150

* P388 leukemia (10 6 cells/mouse) was inoculated (i.p.) on day 0. CY (100 m g / k g ) was administered (i.p.) on day 1. * The schedule for pyrimidinone administration, (i.p.) seven injections on days 2, 6, 10, 14, 18, 22, and 26 two injections on days 6 and - 2 nine injections on days 6, - 2 , 2, 6, 10, 14, 18, 22, and 26.

Table 2. Effect of dosing frequency of pyrimidinone administration*

Treatment Sterile vehicle 100 CY + vehicle ABPP CY + A B P P

ABMFPP CY + A B M F P P

AIMFPP CY + A1MFPP

Dose (mg/kg/injection) CY Pyrimidinone 0 100 0 100 100 100 100 0 100 100 100 100 0 100 100 100 100

0 0 200 200 200 200 200 125 125 125 125 125 100 100 100 100 100

Frequency of pyrimidinone administration

1×

3x

1× 3x 5× 7× 1× -3× 1× 3× 5× 7× 1×-3× 1× 3× 5x 7×

Median death (day)

ILS (°7o)

10 21.5 11 12 28 27 28 37 1 0 . 5 - 11.5 32 29 29.5 35.5 11 11.5 30 30 28.5 29

115 10 20 180 ~ 170~ 180 + 270* 5 15 220* 190 ¢ 195' 255* 10-15 200* 200 185* 190 ¢

*Tumor (106 cells/mouse) was inoculated (i.p.) on day 0 and CY was injected (i.p.) on day 1. SV-100 or pyrimidinone was injected (i.p.) on day 2 and every 4 days thereafter for a number of injections as indicated. * P < 0.05 F-test for synergy based on the comparison of mean life span of 50-day non-survivors. * P < 0.01.

longer statistically significant when ABMFPP was g i v e n 8 d a y s a f t e r C Y a d m i n i s t r a t i o n (Fig. 2). I n a n a t t e m p t to d e t e r m i n e w h e t h e r C Y h a s a n y effect on the pharmacological behavior of p y r i m i d i n o n e , a d o s e o f A B M F P P (125 m g / k g ) w a s given immediately after the CY administration. D e t e r m i n a t i o n o f t h e d r u g level in s e r u m (Fig. 3) c l e a r l y i n d i c a t e s t h a t C Y d i d n o t a p p r e c i a b l y alter t h e kinetics of absorption and excretion of ABMFPP w i t h a t~ v a l u e o f a p p r o x i m a t e l y 2 1 / 2 h. C Y also d i d

n o t c a u s e a n y a l t e r a t i o n in t h e m e t a b o l i c p a t t e r n o f A B M F P P , s i n c e a l m o s t all t h e d r u g in t h e s e r u m w a s in u n c h a n g e d f o r m ( d a t a n o t p r e s e n t e d ) .

Influence o f schedule o f pyrimidinone treatment W h e n e i t h e r A B M F P P or A I M F P P w a s g i v e n 6 a n d 2 d a y s p r i o r to t h e t u m o r i n o c u l a t i o n , t h e r e w a s no apparent preventive effect against tumor d e v e l o p m e n t . W h e n u s e d in c o m b i n a t i o n w i t h C Y ,

35

Pharmacological Evaluation of Pyrimidinones

exudates were determined (Fig. 4). The half-life (t,~) of ABMFPP in peritoneal exudates was approximately 4 h, which is about twice that in serum. A drug concentration of 6.5 ~g/ml was still present in the peritoneal exudate 24 h after the drug administration.

A8MFPP (125mg/kg, i.p.) • Serum

• Peritoneal exudate

25

20

Effect of dosing administration

15

51 Q

~

~

;2

;6

2'o

2~

Hour

Fig. 4. Determination of levels of ABMFPP in serum and peritoneal exudates of BDF~ mice. After a single i.p. injection of ABMFPP (125 mg/kg). The serum or peritoneal wash samples were collected and pooled from three mice at the time indicated.

namely 7 and 3 days prior to CY administration, these pyrimidinones yielded no better therapeutic effect than the treatment with CY alone (Table 1). Furthermore, there was little, if any, antitumor activity when animals received two doses before and seven doses of pyrimidinone after the tumor inoculation. Using a total of nine doses of pyrimidinone in conjunction with CY was no better than using a seven dose injection schedule following CY administration (Table 1).

Effect of dosing frequency administration

of pyrimidinone

In order to determine the minimum dosing frequency achieving a significant synergism with CY, either ABPP, ABMFPP or A I M F P P at their respective optimal dosage, was given starting on day 2 and every 4 days thereafter for a maximum of either 1, 3, 5 or 7 injections (Table 2). A statistically significant effect (P < 0.05 or P < 0.01) was observed with all of the three pyrimidinones used with an ILS ranging from 180 to 220% as compared to a 115% ILS for the animals that received CY treatment alone. For some pyrimidinones such as ABPP, multiple injections appeared to be superior to a single injection of the agent (Table 2). After a single i.p. injection of ABMFPP (125 mg/ kg), the drug levels in both serum and peritoneal

interval

of

pyrimidinone

For the purpose of determining the optimal interval between pyrimidinone dosing, either ABPP, ABMFPP or AIMFPP was given 1 day after CY administration. The subsequent injections were given at 4 day, 7 day, I0 day or 14 day intervals for a maximum of 7, 4, 3 or 2 injections, respectively. ABMFPP or AIMFPP given at intervals from 4 to 14 days between dosings yielded a statistically significant synergism with CY against P388 leukemia (Table 3). ABPP appeared to be less effective when given at a 14-day interval for a total of two injections. When comparing the administration of ABMFPP at a 2 day interval to that at a 4 day interval, the latter appeared to be superior (Li et al., 1984). With the 2 day interval of administration, the serum level of the second injection of the pyrimidinone, such as ABPP, was similar to that of the first injection with a half-life of approximately 21A h. Only a very low drug level (0.5 [ag/ml) was detected 24 h later (data not presented).

Assay of the cytotoxicity of NK cells after ABPP administration The activity of NK cells of peritoneal exudates, expressed as percent of cytotoxicity was determined after the mice received either a single i.p. injection or four weekly injections of ABPP (100 mg/kg/ injection) alone or in combination with CY. The results (Fig. 5) indicate that even with a single injection, ABPP markedly stimulated the cytotoxicity of NK cells against YAC-I target cells. The stimulation reached a maximum at around day 5 and returned to the baseline at the end of 19 days. The multiple injections of ABPP also produced a maximum stimulation of NK ceil activity around day 5 and the stimulation decreased gradually thereafter; however, a significant level of NK cell cytoxicity (> 30%) was still observed at the end of 26 days. When used in combination with CY, the cytotoxic agent neither caused any apparent delay of NK cell stimulation by ABPP under the experimental conditions nor altered the magnitude of the stimulation (Fig. 5).

L. H. Ll et al.

36

Table 3. Effect of dosing interval of pyrimidinone administration Treatment

Pyrimidinone administration Dose Interval* No. of (mg/kg/injection) (day) injections*

CY (mg/kg)

Sterile vehicle 100 CY + vehicle

0 100

0 0

ABPP CY + A B P P

0 100 100 100 100

100 100 100 100 100

4

14 4 7 10 14

2

ABMFPP CY + A B M F P P

0 100

125 125

4 - 14 4

2

AIMFPP CY + A I M F P P

0 (1

ILS %

30-Day Survivors

0* 100

0/8 0/8

7

0--5 190~ 210 ~ 200 ~ 150'

0/8 3/8 5/8 3/8 2/8

7

0 20 200 ~ 220 ~ 170" 2351

0/8 4/8 6/8 2/8 7/8

7

0 - 10 195 ~ 185 ~ 180 ~ 190 ~

0/8 3/8 3/8 2/8 4/8

0 0

7 4 3 2

7

100

125

7

4

100 100

125 125

10 14

3 2

0 100 100 100 100

100 100 100 100 100

4-14 4 7 10 14

2 7 4 3 2

*Tumor (106 cells/mouse) was inoculated (i.p.) on day 0 and CY was injected (i.p.) on day 1. SV-100 or pyrimidinone was injected (i.p.) on day 2 and the injections thereafter were indicated in the table. + The median death of vehicle treated mice was 10 days (ranges 9 - 11 days). * P < 0.05 F-test for synergy based on the comparison of mean life span of 40-day non-survivors. " P < 0.01.

0 ABPP (lx)

Z~ CY + A B P P ( l x )

•

•

ABPP (4x)

Oral administration o f pyrimidinone

CY + A B P P ( 4 x )

80 -,L

o

60

o

'~ 40

4O

zo

20

,,=, 10

20

30

10

20

30

DAY

Fig. 5. Effects of CY a n d / o r A B P P on NK cell activity of mouse peritoneal exudates. Tumor-free BDFL mice received a single injection (i.p.) of CY (100 m g / k g ) on day 1 and A B P P (100 m g / k g ) on day 2 a n d / o r weekly injections on day 9, 16 and 23 (arrows). The peritoneal exudates were collected on day 5, 12, 19 and 26 and were prepared for assaying NK cell cytotoxicity, (%) against YAC-1 cells. The assay procedure is described in the text. [] Sterile vehicle 100; • CY alone; O A B P P alone, single injection; e A B P P alone, 4 injections; ACY plus A B P P (single injection); • CY plus A B P P (four injections).

ABPP was given orally starting on day 2 and every 4 days thereafter for a maximum of seven a d m i n i s t r a t i o n s . O n l y at 600 m g / k g / a d m i n i s t r a t i o n d i d A B P P yield a s t a t i s t i c a l l y s i g n i f i c a n t s y n e r g i s t i c e f f e c t ( P < 0.05) w i t h C Y a g a i n s t P 3 8 8 l e u k e m i a a n d six o u t o f e i g h t m i c e s u r v i v e d l o n g e r t h a n 30 d a y s . T h e e f f e c t w a s less c e r t a i n w h e n a l o w e r d o s e (200 mg/kg/administration) was used (data not presented). I n a n o t h e r e x p e r i m e n t , A B P P w a s g i v e n at a 2 d a y interval for a maximum of either seven or fourteen a d m i n i s t r a t i o n s in c o m p a r i s o n to t h e s t a n d a r d protocol of a 4 day interval for a maximum of seven a d m i n i s t r a t i o n s . A t 200 m g / k g , t h e c o m b i n a t i o n effect with CY was not significantly different statistically from the therapeutic effect of the treatment with CY alone whether the mice received s e v e n or f o u r t e e n d o s e s o f A B P P ( T a b l e 4), a l t h o u g h t w o o u t o f e i g h t m i c e s u r v i v e d l o n g e r t h a n 30 d a y s w i t h t h e 14-day t r e a t m e n t . H o w e v e r , at t h e s a m e dosage, the combination effect was statistically s i g n i f i c a n t ( P < 0.01) w h e n A B P P w a s g i v e n at a 4 d a y i n t e r v a l . T h e s e r e s u l t s collectively s u g g e s t t h a t

37

Pharmacological Evaluation of Pyrimidinones Table 4. Combination therapy of P388 leukemia with cyclophosphamide and oral administration of pyrimidinone Number of ABPP administration

Treatment*

Sterile vehicle 100 ABPP, every 4 days 200mg/kg 800mg/kg ABPP, every 2 days 200mg/kg 800mg/kg

Vehicle or pyrimidinone alone ILS* 30-day (%) survivors

CY + pyrimidinone ILSt 30-day (%) survivors

0

0/8

95

0/8

7 7

5 11

0/8 0/8

163' 216'

2/8 5/8

7 14 7 14

16 5 21 16

0/8 0/8 0/8 0/8

111 116 184' 184'

0/8 2/8 2/8 3/8

* Tumor (10 6 cells/mouse) was inoculated (i.p.) on day 0 and CY was injected (i.p.) on day 1. SV-100 or pyrimidinone was administered (p.o.) on day 2 and every 2 or 4 days thereafter for a maximum of seven or fourteen administrations. * Median death (day) of untreated control was 9.5 days. * P < 0.01, F-test for synergy based on the comparison of mean life span of 40-day non-survivors.

the dosage of 200 mg/kg/administration of ABPP appeared to be too low to ensure a beneficial effect when used in combination with CY. AT 800 m g / k g / administration, ABPP produced a statistically significant synergism ( P < 0.01) with CY whether the mice received a maximum of seven or fourteen administrations of the agent at a 2 day interval. However, the effects were not better than treatment with ABPP given at a 4 day interval. In the latter case, a 216% ILS (P < 0.01) was observed with the combination therapy as compared to a 95% ILS with CY treatment alone. Five out of eight mice survived longer than 30 days (Table 4). The oral administration of either A B M F P P or A I M F P P has not been clearly demonstrated to be beneficial with CY (data not presented).

DISCUSSION Three pyrimidinones, namely ABMFPP, A I M F P P and ABPP, were chosen for this study. ABPP is currently under clinical investigation. A B M F P P was studied intensively because of its initially favorable therapeutic effect against P388 leukemia when compared to that of ABPP (Li et al., 1984); however, these three pyrimidinones were found to produce comparable results against a number of experimental tumors in a subsequent and more comprehensive study (Li et al., 1985). Hence, the results obtained with A B M F P P would generally

be applicable to results obtained with other pyrimidinones. This study was undertaken to evaluate the pharmacological behaviors of these pyrimidinones in an attempt to optimize conditions for the maximum therapeutic effect against P388 leukemia when used in conjunction with CY, since none of these pyrimidinones by themselves were active against this tumor (Table 2). Since the gap between the administration of the cytotoxic drug and immunomodulators or biological response modifiers may have a marked influence on the outcome of the combination therapy, the optimal gap between CY and pyrimidinone administration was studied at the onset of this investigation. The concomitant drug administration was found to be no better than the sequential administration, and the best therapeutic result was observed when the pyrimidinone was given one day after CY administration (Fig. 2). The combination effect was no longer clearly synergistic when the pyrimidinone was given 8 days after CY administration. It appears reasonable to speculate that the rather close gap between CY and pyrimidinone administration would effectively reduce the chance for the repopulation of residual tumor cells after CY treatment. The optimal gap may vary with different tumors, particularly those with different growth characteristics. Treatment with pyrimidinone prior to the P388 leukemia inoculation did not improve the host resistance to the tumor (Table 1). This might be in part related to the relatively high tumor inoculation

38

L.H. LI et al.

(10 6 cells/mouse). Treatment with pyrimidinone prior to CY administration yielded no better therapeutic value than the treatment with CY alone. A statistically significant synergism was observed even with a single i.p. injection of either ABPP, ABMFPP or AIMFPP 1 day after CY administration. It appeared that multiple injections of some pyrimidinones, such as ABPP, produced a better therapeutic effect (Table 2). The pyrimidinone level was about twice as high in the peritoneal cavity where the drug suspensions had been inoculated as in the serum (Fig. 4). The absorption and clearance of pyrimidinone, based on the serum level determination, was apparently not affected by CY administration (Fig. 3). A statistically significant synergism (P < 0.01) with CY was obtained whether the interval between the first dose and subsequent dose(s) was 4, 7, 10 or 14 days (Table 3). A great number of long term (> 30 day) survivors were recorded as compared to none with CY treatment alone. Again, the combination effect appeared to be less dramatic when ABPP was given 14 days apart for a total of two doses (Table 3). In general, this study clearly demonstrates that the beneficial effect of the combination therapy can be accomplished without using intensive schedules of pyrimidinone administration and a weekly schedule would be sufficient with this tumor and several other experimental tumors (Li et al., 1986). Possibly, the schedule of administration may vary with other experimental and/or clinical tumors. Although a single injection of pyrimidinone could produce a significant therapeutic improvement with CY, it would be advantageous to use multiple administrations of the pyrimidinones to maintain the maximal therapeutic effect. Since we have shown that the synergistic antitumor effect appeared, at least in part, to be related to the initial tumor burden reduction by CY and the marked enhancement of both NK cell and macrophage activities by the pyrimidinone (Li et al., 1985), NK cell activity of BDF~ mice was determined after single or multiple injections of ABPP alone and in combination with CY. A significant stimulation of NK cell cytotoxicity against target tumor cells was observed and the stimulation reached a maximum around 3 days after a single injection of ABPP. The activity returned to the basal level at the end of 19 days (Fig. 5). With multiple injections, the stimulation of NK cell activity decreased gradually with subsequent ABPP injections, although

significant levels of NK cell activity were maintained during the course of experimentation. In both cases, CY neither caused a delay in, nor altered the magnitude of stimulation of NK cell activity by ABPP (Fig. 5). The significance of these observations may be in part reflected by the fact that the multiple injections were superior to the single injection of ABPP in combination with CY against P388 leukemia and resulted in a great number of long-term survivors (Tables 2 and 3). Also, the initial and profound stimulation of NK cell activity after animals received a single injection of ABPP appeared to be critical and sufficient to produce a significant synergism with CY. Although intraperitoneal administration of immunomodulators appeared to be effective in the treatment of clinical cancers such as bladder and ovarian after chemotherapy (Berek, Knapp, Hacker, Lichtenstein, Jung & Spina, 1985), it would be more desirable for immunomodulators, such as pyrimidinones, to be effective orally against other kinds of tumors. As discussed in the previous sections, this type of agent requires multiple or chronic administrations in order to assure maximal therapeutic value. Also, it would be more convenient to use this type of agent clinically on an outpatient basis when patients are in remission after receiving the initial treatment with surgery and/or chemotherapy. It is rather satisfying to see that the combination therapy with CY was highly synergistic statistically when ABPP was given orally. The synergistic antitumor effect was comparable whether ABPP was given every 4 days for a maximum of seven administrations or every 2 days for a total of seven or fourteen administrations (Table 4). In general, our results indicate that although pyrimidinones do not require an intensive schedule of administration, some pyrimidinones, such as ABPP, require multiple administrations for their maximum activity whether they are given intraperitoneally or orally. The therapeutic value of the oral administration of ABPP against experimental tumors appeared to be in concert with the clinical finding reported recently by Earhart, Hamilton, Henry, Hanover, Maile, Agrawal & Todd (1985). A c k n o w l e d g e m e n t s - - We thank Mrs. J. Moerman, Mr. G.

D. Kramer and Mr. A. Tomilo for their skillful technical assistance, and Mrs. Ann Kiewiet for her help in preparation of this manuscript.

Pharmacological Evaluation of Pyrimidinones

39

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