EXPERIMENTAL PARASITOLOGY
40, 95-102 ( 1976)
Plasmodium berghei: Combining Folic Acid Antagonists for Potentiation against Malaria infections in Mice
Dikion
of Medicinal
KENNETH
E.
Chemistry,
Walter Reed Army Institute
Washington,
KINNAMON'
DC.
20012,
of Research,
U.S.A.
AND ARBA L. ACER L’AND ROBERT W.
ORCHARD 3
Department of Parasitology, School of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, U.S.A.
(Accepted KINNAMON,
for publication
29 September
1975)
KENNETH E., AGER, ARBA L., AND ORCHARD, ROBERT W.
1976. Plasmodium
berghei: Combining folic acid antagonists Experimental Parasitology 40, 95-102.
for potentiation against malaria infections in mice. The drugs 2,4-diamino-6-( 2-naphthyl-sulfonyl)quinazoline (WR 158,122) and its tetrahydro analog (WR 180,872) were found to act synergistically in reducing the parasitemia of Plasmodium berghei infected mice. Evidence was found that both the drugs which are potent folic acid antagonists were acting at different sites in the folic acid metabolic pathway, neither of which was at the action site of p-aminobenzoic acid (PABA) inhibitors. Advantages of drug combinations in malaria chemotherapy are discussed. INDEX DESCRIPTORS: Antifols; Chemotherapy, malaria; Combined drug therapy; Dihydrofolate reductase inhibitors; Drug combinations; Drug synergism; Plasmodium berghei; Mice; Drug antagonism; Inhibitors, metabolic; Malaria; Metabolic inhibitors; Enzymes; p-Aminobenzoic acid; Folic acid.
The advantages of combining drugs in the treatment of malaria are recognized (Schmidt 1973; Thompson and Werbel 1972). Superior activity against drug-sensitive parasites, broader action against drug-resistant strains, less likelihood of permitting the acquisition of resistance 1 Present address: Uniformed Services University of the Health Sciences, 6917 Arlington Road, Bethesda, Maryland 20014, U.S.A. 2 Present address: The Leo Rane Research Laboratory, The University of Miami, Miami, Florida 33142, U.S.A. 3 Present address : Department of Medicinal Chemistry, School of Pharmacy, The University of Georgia, Athens, Georgia 30602, U.S.A.
1976by AcademicPress, Inc. COPyrbm All rightso? reproductionin any form reserved.
than when the components are used alone, and lower host toxicity are benefits noted. The first demonstration of the potentiating effects of two such drugs acting at two different sites in the folic acid metabolic pathway was shown by Greenberg et al. in 1948. Sulfadiazine and proguanil were shown to be synergistic in their action against Plasmodium gallinaceum. Points A and B in Fig. 1 show the sequential blocking sites, respectively, for the p-aminobenzoic
acid
(PABA)
competitor,
sulfa-
diazine, and the dihydrofolate (DHF) reductase inhibitor, proguanil. Since that time several combinations of drugs believed to be
acting
at these
two
blocking
sites
96
KINNAMON,
AGER
AND
ORCHARD
have been shown to act synergistically. It may be reasoned that a substance acting to block at yet another point in the meta-
bolic pathway may potentiate antimalarial drugs acting at either A or B in Fig. 1. 2 Such is the case for the thymidylate syn@Jso*$$NH, WR ‘58,‘22 thetase inhibitor S-fluoro-2’deoxyuridine which has been shown to act synergistically with either a PABA competitor or a DHF 2. 4-Dlamlna-6-(2-Naphihyl-Sulfonyli-Oulnazoline reductase inhibitor in reducing the growth of E. co& (Then 1974). Use of lethal tetrahydro-derivatives to act at Point C (Fig. 1) in lieu of searching 2, 4- Dlamlno-6-i2-NophfhyI-Sulfonyl)-5.6,7,8for new inhibitors of DHF reductase in Tetr(lhydroqu,nazol,ne cancer chemotherapy has been proposed FIG. 2. Drugs used in this investigation. (Friedkin et al. 1971) . The present work was conducted to determine if such potentiation could be demonstrated against ma- WR 158,122 and WR 180,872 are potent laria infections of mice. folic acid antagonists as determined in bacterial systems (Smith and Genther 1972). MATERIALS AND METHODS WR 158,122 (Elslager and Werbel 1974) Drugs Selected and WR 180,872 (Shapiro, unpublished) In this study, drugs selected for action were synthesized under U.S. Army conat Points A and B (Fig. 1) were respec- tracts. The structures of these three comtively sulfadiazine and the DHF reductase pounds are shown in Fig. 2. inhibitor
2,4-diamino-6-
(2-naphthyl-sulfo-
nyl)-quinazoline (hereafter designated WR 158,122). The third compound selected (designated WR 180,872) was the tetrahydro analog of WR 158,122 which we reasoned might act at Point C (Fig. 1). Both WBA -A
INTERFERENCE
POINT
B INTERFERENCE
POINT
C INTERFERENCE IN5.‘Oa THF DEPENDENT PRODUCTS
POINT
I
FOLlC ACID I DHF lTHF
N5, N”,
I ONE CARBON UNIT TRANSFER REACTIONS
FIG. 1. Interference with folic acid dependent reactions. Abbrevations : PABA = p-aminobenzoic acid; DHF =dihydrofolate; THF = tetrahydrofolate. , N6 > N’” N”, lo THF = NE formyl THF; N6 methyl THF, A’ formimino THF, N”’ formyl THF, N”, ” methenyl THF, N5. ‘” methylene THF.
Animals and Drug Administration Carworth CF-1 female mice, each weighing 20-24 g were used. They were fed a standard pelleted laboratory chow, except in PABA experiments in which the food was ground. PABA was mixed with feed scheduled for mice to receive the PABA supplement. Test compounds were administered twice daily in aqueous 0.5% hydroxyethylcellulose-0.1 s Tween 80 solution after ultrasonification in a volume of 10 ml/kg. Administration was begun on the third day after parasite inoculation and continued for three consecutive days. Malaria Infections Mice were infected with the KBG 173 strain of Plasmodium berghei by intraperitoneal administration of 2.5 x 10” parasitized erythrocytes. Infections were assessed from Giemsa stained blood films made 1 day after completion of the treatment. Approximately 200 erythrocytes wer el
FOLIC
ACID
ANTAGONIST
POTENTIATION
examined when as many as 5% were parasitized. In lighter infections, examination was continued until 10 parasites were counted and at least 10 fields were examined. If fewer than 10 parasites were found 100 fields were examined. The percentage of suppression of parasites in mice was calculated for each treated animal relative to the mean values in the controls; from these the mean percentage of suppression was computed. Doses required for a particular degree of effect were read from probit-log graphs. Assessment of PABA Antagonism Drug Synergism
and
PABA antagonism. The test for antagonism by p-aminobenzoic acid (PABA) was conducted as previously described (Thompson 1972). Mice were divided into groups of 7 mice each and infected. On the same day the mice were weighed and put on a diet of 0.25% PABA. Drugs were administered orally by gavage. Drug synergism. The test for drug synergism was as previously described by Thompson et al. (1969) and was somewhat similar to the method discussed by Jacobs et al. ( 1963). Drugs were administered subcutaneously in all experiments, except the one in which the sulfadiazine and WR 158,122 assessment was made. In the latter the compounds were administered orally. The test is based upon the suppression by 90% or 70$>, i.e., SD!,0 or SDTo of the compounds alone and of the mixtures and were estimated by plotting percentage of parasitemia suppression on a probit scale against dose on a logarithmic scale. In plotting the parasitemia scale, five points, seven animals per point, ranging from a suppression of 6 to loos, were used per drug or combination of drugs in each test. An SDno was used in each instance unless 90% suppression was not attained with one or more treatments. The analyses for synergism were based upon a partitioning of the response, R, into fractional amounts of each of the two drugs
IN
MALARIA
97
THERAPY
of the mixture according to each drug’s contribution to R. The value R was based upon the response observed, e.g., SDBo, when each drug was given alone. That is, suppose dose x of drug A alone and dose y of drug B alone are selected so that the same response R is obtained. Similarly, drug A may be combined at the appropriate fractional dose level cx with drug B at the complementary fractional dose level ( l-c) y so that the same response R is obtained. If the combination cx of drug A and (l-c) y of drug B produces R then the combination is simply additive; the expected fractional contribution of R is assumed to be 0.5 for each of the two drugs at the respective proper dose level, although the A to B ratio may be dissimilar. If dose levels of A and B in combination are less than expected to obtain R, then the combination is synergistic (greater than TABLE Parasit,emia Suppression Infected Mice after Three with and wit,hout PABA o.02.Y3J.
I in Plasmodium berghei Ijays of Chemotherapy at a Dietary Level of
Percent,age suppression Drug alone
of
Drug + PABA
Sulfadiazine
2.0 1.0 0.5 0.125
99.9 98.2 85.2 19.1
10.2 19.2 10.2 16.9
WII.
158,122
2.56 0.64 0.16 0.04
99.3 60.1 19.9 8.9
90.1 62.9 16.5 11.1
WR
180,872
2.56 0.64 0.16 0.04
99.9 58.9 29.2 12.8
99.9 64.4 57.7 19.8
a On the basis of previous experiments the highest doses selected were known to give approximately 99% suppression, ot’her doses selected were multiples of 2 or 4 known to give the 4th and lowest level of IO-207, suppression.
98
KINNAMON,
AGER
TABLE Data Analysis of Joint Antimalarial sitemia of Plasmodium berghei Infected the SDro. Drugs
Action Mice,
AND
ORCHARD
II
of WR 158,122 or WH Based upon ParCtioning
180,872 and Sulfadiazine upon Paraof Mixtures in Terms of Fractions of
SD,o” observed hdkdday)
Drug
Expected additive 1. Sulfadiazine 2. WR 158,122 3. 3 Parts sulfadiazine 1 Part WR 158,122
0.15 0.34 0.033 0.011
if
fractions effecting
of mixtures an SD,0
Found
Expected/ found
0.5 0.5
0.22 0.032
2.27 15.63
0.5 0.5
0.507 0.022
0.99 22.7
0.5 0.5
0.74 0.011
0.67 45.5
0.5 0.5
0.631 0.044
0.792 11.364
0.5 0.5
0.446 0.108
1.121 4.630
0.5 0.5
0.096 0.278
5.208 1.799
0.044 4. 10 Parts sulfadiazine 1 Part WR 158,122
0.076 0.0076 0.0836 0.1113 0.0037
5. 30 Parts sulfadiazine 1 Part WR 158,122
0.1 I.50 0.26 0.36 0.164 0.016
6. Sulfadiazine 7. WR 180,872 8. 10 Parts sulfadiazine 1 part WR 180,872
0.18 9. 3 Parts sulfadiazine 1 part WR 180,872
0.116 0.039 0.155 0.025 0.100
10. 1 Part sulfadiazine 4 Parts WR 180,872
0.125 a SD70
= That
dose causing
a 70y0
suppression
of parasitemia.
additive). Conversely, if dose levels of the two drugs are required in amounts that are more than expected to obtain R then the combination is antagonistic (less than additive). See “Synergism: Sulfadiazineantifols” under Results for an example which serves to illustrate the analyses. RESULTS
nificantly prevented the suppression effected by sulfadiazine treatment. At the top three dose levels there was a greater than fivefold inhibition of parasitemia suppression There was no similar inhibition of parasitemia suppression in mice treated with either the 2,4-diaminoquinazoline WR 158,122 or its tetrahydro analog, WR 180,872.
PABA Antagonism Results of PABA antagonism assessment are shown in Table I. Inspection reveals that PABA at a dietary level of 0.025% sig-
Synergism:
Sulfadiazine-antifols
Initial synergism experiments were conducted to determine the combined effects
FOLIC
ACID
ANTAGONIST
POTENTIATION
of either WR 158,122 or WR 180,872 with sulfadiazine. The analyses for synergism are shown in Table II. For clarification, a more detailed explanation of a part of Table II is appropriate. The SD50 values of 0.15, 0.34-, 0.044-, 0.0836, and 0.1150 mg/kg/day (MKD) were found by inspecting the dose response curves respectively for sulfadiazine, WR 158,122, and the 3:1, 1O:l and 3O:l sulfadiazine-WR 158,122 combinations. Using these SDTO values and the appropriate ratios, the SDTo values for the components of each mixture were calculated, e.g., the 0.044 MKD figure for the 3:l ratio yields, respectively, 0.033 and 0.011 MKD (0.033 + 0.011 = 0.044). The expected fractional values if the mixtures were simply additive would be the total of one, i.e., 0.5 each for the two drugs in the mixtures. The expected fractional values are shown in the third column of Table II. The SD,, value of the mixture components calculated from fractional values actually found are shown in the fourth column. These “found” values were calculated by dividing the SD70 value found in the mixture SDTo by the dose rate level value found when the compound was given alone, e.g., in the 3:l combination. The sulfadiazine found fractional value shown in Column 4 is 0.22 (0.033 MKD/0.15 MKD = 0.22). The expected to found ratios are shown in the last column and are obtained by dividing the expected fractional value in Column 3 by the corresponding found fractional value in Column 4. In the case of the 3:l ratio, the expected to found ratios are 2.27 and 15.63 for sulfadiazine and WR 158,122, respectively. The best combination may be observed by noting in Column 2 which MKD is smallest. Inspection of Table II, Column 2 reveals that combination to be 3:I. Other analyses are similarly conducted. A graphical representation of the values obtained in TabIe II is shown in Fig. 3. The use of this type diagram, introduced by Loewe and described by Gaddum
IN
MALARIA
99
THERAPY
AI 4 More Than
0
0.2
Addltlve
Acllon
0.4
Proportional WR 158,122
0.6
Amount
(Potanttati
08 of on SD,,
IO of
or WR 180,872
FIG. 3. Loewe diagram illustrating the degree of potentiation produced in effecting 70% suppression of parasitemia of P. berghi infected mice by mixing WR 158,122 or WR 180,872 and sulfadiazine for 3 day treatment. The positions of the combination points below the line of addition are evidence of potentiation.
(1959), is obtained by plotting the found fractional values in Table II, Column 4. The Fig. 3 diagram represents the combination of sulfadiazine and either WR 158,122 or WR 180,872. The x axis represents the proportional amount of sulfadiazine which effects an SDTOin the absence of any other drug. Similarly, the y axis represents the proportional amount of WR 158,I22 or WR 180,872 to produce an SD70 when it is administered alone. If a combination of sulfadiazine and either WR 158,122 or WR 180,872 were administered and the found fractional value from Table II, Column 4 were such that it fell on the hypotenuse the conclusion would be that the drugs in that particular combination were simply additive in their effects. Further, if an SDTo were produced by a drug combination that when plotted fell below the hypotenuse, the conclusion would be that the drug combination was more additive, i.e., potentiation existed. On the other hand, if when treating, presence of WR 158,122 or WR 180,872 necessitated the presence of an increased quantity of sulfadiazine to produce an SD,,; or conversely if the presence of sulfadiazine required increased quan-
100
KINNAMON,
AGER
TABLE Data
Analysis of Joint Antimalarial berg&i Infected Mice, Based
Action of WR upon Partitioning
AND
OHCHARD
III
158,122 and Wlb 180,872 of Mixtures in Terms
SDd
Drugs
Drug
observed (mg/k/day) Expected additive 1. WR 158,122 2. WR 180,872 3. 1 Part WR 158,122 1 Part WR 180,872
0.88 1.1 0.325 0.325
if
upon I’arGlemis of Plasmodium of Fractions of the SD90 fractions effecting Found
of mixtures an SD90 Expected/ found
0.5 0.5
0.369 0.295
1.355 1.695
0.5 0.5
0.318 0.127
1.572 3.937
0.5 0.5
0.373 0.075
1.340 6.667
0.3 0.0c
0.403 0.032
1.241 15.625
0.5 0.5
0.248 0.456
2.016 1.097
0.5 0.5
0.218 0.800
2.294 0.625
0.5 0.5
0.104 0.947
4.808 0.528
0.5 0.5
0.045 1.042
11.111 0.480
0.5 0.5
0.472 0.018
1.059 27.778
0.65 4. 2 Parts WI1 158,122 1 Part WR 180,872
0.28 0.14
0.42 5. 4 Parts WR 158,122 1 Part WR 180,872
0.328 0.082 0.41
6. 10 Parts WR 158,122 1 Part WR 180,872
0.3546 0.0355 0.30
7. WR 158,122 8. WR 180,872 9. 1 Part WR 158,122 2 Parts WR 180,872
1.1 1.2 0.273 0.547 0.82
10. 1 Part WR 158,122 4 Parts WR 180,872
0.24 0.96 1.2
11.
1 Part WR 158 122 10 Parts WR 186,872
0.114 1.136 1.35
12.
1 Part WR 158,122 25 Parts WR 180,872
0.05 1.25 1.3
13. 25 Parts WR 158,122 1 Part WR 180,872
0.519 0.021 0.54
0 SD,,
= That
dose causing
a 90%
suppression
of parasitemia.
tities of WR 158,122 or WR 180,872 to produce an SD,, the conclusion would be that the drug combination was less than addi-
tive, i.e., antagonism existed. When points from such less than additive combinations 1 are plotted on a diagram such as in Fig. 3 i
FOLIC
ACID
ANTAGONIST
POTENTIATION
IN
MALARIA
101
THERAPY
the points
appear above the hypotenuse. It may be noted from Fig. 3 that all points from the sulfadiazine-antifol combinations used in the present study fell well below the line of addition. Synergism:
WR 158,122-WR
180,872
Combinations of the 2,4-diaminothioquinazoline, WR 158,122 and its tetrahydro analog, WR 180,872, were found to be synergistic in all ratios where WR 180,872 made up no more than two-thirds of the drug-combination. The data are summarized in Table III and Fig. 4. The 2:1, 4:1, and 1O:l ratios were most effective; the 25:l ratio less effective but slightly more potentiating than the 1: 1 and 1:2 ratios. The 1:4, 1: 10, and 1:25 ratios were approximately additive in effect. DISCUSSION
In this paper we present evidence that subsequent to treatment of 2’. herghei infected mice with combined doses of different antifolates there is a greater than additive chemotherapeutic effect of the two drugs. Thus, the drugs 2,4-diamino-6- ( 2naphthyl-sulfonyl )-quinazoline ( WR 158,122) and its tetrahydro analog (WR 180,872) are presumed to be acting synergistically by affecting two different sites in the folic acid metabolic pathway, analogous to potent&ion observed when sulfonamides and DHF reductase inhibitors are combined. Further, both of these sites of action are believed to be at a location other than Point A (Fig. 1) for two reasons. First sulfadiazine acts synergistically with either WR 158,122 or WR 180,872. Second, the activity of sulfadiazine is reversed by PABA, whereas the activity of the other two drugs is unaffected by PABA. Since in the metabolic pathway (Fig. 1) there is the conversion from dihydrofolate to tetrahydrofolate, one may suspect on the basis of closer structural similarity that the quinazoline WR 158,122 is active at Point B, whereas the tetrahydroquinazoline WR 180,872 is likely to be more
Proportuxwt
Amount of
of
an
SDgO
WR 180,872
FIG. 4. Loewe diagram illustrating the degree of potentiation produced in effecting 90% suppression of parasitemia of P. berghi infected mice by mixing WR 158,122 and WR 180,872 for 3 day treatment. The positions of the combination points below the line of addition are evidence of potentiation.
active at Point C. It is possible that one or both of these compounds is partially active at both sites. It has been pointed out that cofactors required for cle nom methyl synthesis in P. berghei include tetrahydrofolic acid and that the use of antifolates as antimalarials may serve to block that reaction as well as other folic acid dependent reactions (Langer et al. 1969) . In mammalian metabolic systems it is known that folate coenzymes are involved as carriers in possibly all reactions involving the transfer of one-carbon units, viz, -CH:
40, 95-102 ( 1976)
Plasmodium berghei: Combining Folic Acid Antagonists for Potentiation against Malaria infections in Mice
Dikion
of Medicinal
KENNETH
E.
Chemistry,
Walter Reed Army Institute
Washington,
KINNAMON'
DC.
20012,
of Research,
U.S.A.
AND ARBA L. ACER L’AND ROBERT W.
ORCHARD 3
Department of Parasitology, School of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, U.S.A.
(Accepted KINNAMON,
for publication
29 September
1975)
KENNETH E., AGER, ARBA L., AND ORCHARD, ROBERT W.
1976. Plasmodium
berghei: Combining folic acid antagonists Experimental Parasitology 40, 95-102.
for potentiation against malaria infections in mice. The drugs 2,4-diamino-6-( 2-naphthyl-sulfonyl)quinazoline (WR 158,122) and its tetrahydro analog (WR 180,872) were found to act synergistically in reducing the parasitemia of Plasmodium berghei infected mice. Evidence was found that both the drugs which are potent folic acid antagonists were acting at different sites in the folic acid metabolic pathway, neither of which was at the action site of p-aminobenzoic acid (PABA) inhibitors. Advantages of drug combinations in malaria chemotherapy are discussed. INDEX DESCRIPTORS: Antifols; Chemotherapy, malaria; Combined drug therapy; Dihydrofolate reductase inhibitors; Drug combinations; Drug synergism; Plasmodium berghei; Mice; Drug antagonism; Inhibitors, metabolic; Malaria; Metabolic inhibitors; Enzymes; p-Aminobenzoic acid; Folic acid.
The advantages of combining drugs in the treatment of malaria are recognized (Schmidt 1973; Thompson and Werbel 1972). Superior activity against drug-sensitive parasites, broader action against drug-resistant strains, less likelihood of permitting the acquisition of resistance 1 Present address: Uniformed Services University of the Health Sciences, 6917 Arlington Road, Bethesda, Maryland 20014, U.S.A. 2 Present address: The Leo Rane Research Laboratory, The University of Miami, Miami, Florida 33142, U.S.A. 3 Present address : Department of Medicinal Chemistry, School of Pharmacy, The University of Georgia, Athens, Georgia 30602, U.S.A.
1976by AcademicPress, Inc. COPyrbm All rightso? reproductionin any form reserved.
than when the components are used alone, and lower host toxicity are benefits noted. The first demonstration of the potentiating effects of two such drugs acting at two different sites in the folic acid metabolic pathway was shown by Greenberg et al. in 1948. Sulfadiazine and proguanil were shown to be synergistic in their action against Plasmodium gallinaceum. Points A and B in Fig. 1 show the sequential blocking sites, respectively, for the p-aminobenzoic
acid
(PABA)
competitor,
sulfa-
diazine, and the dihydrofolate (DHF) reductase inhibitor, proguanil. Since that time several combinations of drugs believed to be
acting
at these
two
blocking
sites
96
KINNAMON,
AGER
AND
ORCHARD
have been shown to act synergistically. It may be reasoned that a substance acting to block at yet another point in the meta-
bolic pathway may potentiate antimalarial drugs acting at either A or B in Fig. 1. 2 Such is the case for the thymidylate syn@Jso*$$NH, WR ‘58,‘22 thetase inhibitor S-fluoro-2’deoxyuridine which has been shown to act synergistically with either a PABA competitor or a DHF 2. 4-Dlamlna-6-(2-Naphihyl-Sulfonyli-Oulnazoline reductase inhibitor in reducing the growth of E. co& (Then 1974). Use of lethal tetrahydro-derivatives to act at Point C (Fig. 1) in lieu of searching 2, 4- Dlamlno-6-i2-NophfhyI-Sulfonyl)-5.6,7,8for new inhibitors of DHF reductase in Tetr(lhydroqu,nazol,ne cancer chemotherapy has been proposed FIG. 2. Drugs used in this investigation. (Friedkin et al. 1971) . The present work was conducted to determine if such potentiation could be demonstrated against ma- WR 158,122 and WR 180,872 are potent laria infections of mice. folic acid antagonists as determined in bacterial systems (Smith and Genther 1972). MATERIALS AND METHODS WR 158,122 (Elslager and Werbel 1974) Drugs Selected and WR 180,872 (Shapiro, unpublished) In this study, drugs selected for action were synthesized under U.S. Army conat Points A and B (Fig. 1) were respec- tracts. The structures of these three comtively sulfadiazine and the DHF reductase pounds are shown in Fig. 2. inhibitor
2,4-diamino-6-
(2-naphthyl-sulfo-
nyl)-quinazoline (hereafter designated WR 158,122). The third compound selected (designated WR 180,872) was the tetrahydro analog of WR 158,122 which we reasoned might act at Point C (Fig. 1). Both WBA -A
INTERFERENCE
POINT
B INTERFERENCE
POINT
C INTERFERENCE IN5.‘Oa THF DEPENDENT PRODUCTS
POINT
I
FOLlC ACID I DHF lTHF
N5, N”,
I ONE CARBON UNIT TRANSFER REACTIONS
FIG. 1. Interference with folic acid dependent reactions. Abbrevations : PABA = p-aminobenzoic acid; DHF =dihydrofolate; THF = tetrahydrofolate. , N6 > N’” N”, lo THF = NE formyl THF; N6 methyl THF, A’ formimino THF, N”’ formyl THF, N”, ” methenyl THF, N5. ‘” methylene THF.
Animals and Drug Administration Carworth CF-1 female mice, each weighing 20-24 g were used. They were fed a standard pelleted laboratory chow, except in PABA experiments in which the food was ground. PABA was mixed with feed scheduled for mice to receive the PABA supplement. Test compounds were administered twice daily in aqueous 0.5% hydroxyethylcellulose-0.1 s Tween 80 solution after ultrasonification in a volume of 10 ml/kg. Administration was begun on the third day after parasite inoculation and continued for three consecutive days. Malaria Infections Mice were infected with the KBG 173 strain of Plasmodium berghei by intraperitoneal administration of 2.5 x 10” parasitized erythrocytes. Infections were assessed from Giemsa stained blood films made 1 day after completion of the treatment. Approximately 200 erythrocytes wer el
FOLIC
ACID
ANTAGONIST
POTENTIATION
examined when as many as 5% were parasitized. In lighter infections, examination was continued until 10 parasites were counted and at least 10 fields were examined. If fewer than 10 parasites were found 100 fields were examined. The percentage of suppression of parasites in mice was calculated for each treated animal relative to the mean values in the controls; from these the mean percentage of suppression was computed. Doses required for a particular degree of effect were read from probit-log graphs. Assessment of PABA Antagonism Drug Synergism
and
PABA antagonism. The test for antagonism by p-aminobenzoic acid (PABA) was conducted as previously described (Thompson 1972). Mice were divided into groups of 7 mice each and infected. On the same day the mice were weighed and put on a diet of 0.25% PABA. Drugs were administered orally by gavage. Drug synergism. The test for drug synergism was as previously described by Thompson et al. (1969) and was somewhat similar to the method discussed by Jacobs et al. ( 1963). Drugs were administered subcutaneously in all experiments, except the one in which the sulfadiazine and WR 158,122 assessment was made. In the latter the compounds were administered orally. The test is based upon the suppression by 90% or 70$>, i.e., SD!,0 or SDTo of the compounds alone and of the mixtures and were estimated by plotting percentage of parasitemia suppression on a probit scale against dose on a logarithmic scale. In plotting the parasitemia scale, five points, seven animals per point, ranging from a suppression of 6 to loos, were used per drug or combination of drugs in each test. An SDno was used in each instance unless 90% suppression was not attained with one or more treatments. The analyses for synergism were based upon a partitioning of the response, R, into fractional amounts of each of the two drugs
IN
MALARIA
97
THERAPY
of the mixture according to each drug’s contribution to R. The value R was based upon the response observed, e.g., SDBo, when each drug was given alone. That is, suppose dose x of drug A alone and dose y of drug B alone are selected so that the same response R is obtained. Similarly, drug A may be combined at the appropriate fractional dose level cx with drug B at the complementary fractional dose level ( l-c) y so that the same response R is obtained. If the combination cx of drug A and (l-c) y of drug B produces R then the combination is simply additive; the expected fractional contribution of R is assumed to be 0.5 for each of the two drugs at the respective proper dose level, although the A to B ratio may be dissimilar. If dose levels of A and B in combination are less than expected to obtain R, then the combination is synergistic (greater than TABLE Parasit,emia Suppression Infected Mice after Three with and wit,hout PABA o.02.Y3J.
I in Plasmodium berghei Ijays of Chemotherapy at a Dietary Level of
Percent,age suppression Drug alone
of
Drug + PABA
Sulfadiazine
2.0 1.0 0.5 0.125
99.9 98.2 85.2 19.1
10.2 19.2 10.2 16.9
WII.
158,122
2.56 0.64 0.16 0.04
99.3 60.1 19.9 8.9
90.1 62.9 16.5 11.1
WR
180,872
2.56 0.64 0.16 0.04
99.9 58.9 29.2 12.8
99.9 64.4 57.7 19.8
a On the basis of previous experiments the highest doses selected were known to give approximately 99% suppression, ot’her doses selected were multiples of 2 or 4 known to give the 4th and lowest level of IO-207, suppression.
98
KINNAMON,
AGER
TABLE Data Analysis of Joint Antimalarial sitemia of Plasmodium berghei Infected the SDro. Drugs
Action Mice,
AND
ORCHARD
II
of WR 158,122 or WH Based upon ParCtioning
180,872 and Sulfadiazine upon Paraof Mixtures in Terms of Fractions of
SD,o” observed hdkdday)
Drug
Expected additive 1. Sulfadiazine 2. WR 158,122 3. 3 Parts sulfadiazine 1 Part WR 158,122
0.15 0.34 0.033 0.011
if
fractions effecting
of mixtures an SD,0
Found
Expected/ found
0.5 0.5
0.22 0.032
2.27 15.63
0.5 0.5
0.507 0.022
0.99 22.7
0.5 0.5
0.74 0.011
0.67 45.5
0.5 0.5
0.631 0.044
0.792 11.364
0.5 0.5
0.446 0.108
1.121 4.630
0.5 0.5
0.096 0.278
5.208 1.799
0.044 4. 10 Parts sulfadiazine 1 Part WR 158,122
0.076 0.0076 0.0836 0.1113 0.0037
5. 30 Parts sulfadiazine 1 Part WR 158,122
0.1 I.50 0.26 0.36 0.164 0.016
6. Sulfadiazine 7. WR 180,872 8. 10 Parts sulfadiazine 1 part WR 180,872
0.18 9. 3 Parts sulfadiazine 1 part WR 180,872
0.116 0.039 0.155 0.025 0.100
10. 1 Part sulfadiazine 4 Parts WR 180,872
0.125 a SD70
= That
dose causing
a 70y0
suppression
of parasitemia.
additive). Conversely, if dose levels of the two drugs are required in amounts that are more than expected to obtain R then the combination is antagonistic (less than additive). See “Synergism: Sulfadiazineantifols” under Results for an example which serves to illustrate the analyses. RESULTS
nificantly prevented the suppression effected by sulfadiazine treatment. At the top three dose levels there was a greater than fivefold inhibition of parasitemia suppression There was no similar inhibition of parasitemia suppression in mice treated with either the 2,4-diaminoquinazoline WR 158,122 or its tetrahydro analog, WR 180,872.
PABA Antagonism Results of PABA antagonism assessment are shown in Table I. Inspection reveals that PABA at a dietary level of 0.025% sig-
Synergism:
Sulfadiazine-antifols
Initial synergism experiments were conducted to determine the combined effects
FOLIC
ACID
ANTAGONIST
POTENTIATION
of either WR 158,122 or WR 180,872 with sulfadiazine. The analyses for synergism are shown in Table II. For clarification, a more detailed explanation of a part of Table II is appropriate. The SD50 values of 0.15, 0.34-, 0.044-, 0.0836, and 0.1150 mg/kg/day (MKD) were found by inspecting the dose response curves respectively for sulfadiazine, WR 158,122, and the 3:1, 1O:l and 3O:l sulfadiazine-WR 158,122 combinations. Using these SDTO values and the appropriate ratios, the SDTo values for the components of each mixture were calculated, e.g., the 0.044 MKD figure for the 3:l ratio yields, respectively, 0.033 and 0.011 MKD (0.033 + 0.011 = 0.044). The expected fractional values if the mixtures were simply additive would be the total of one, i.e., 0.5 each for the two drugs in the mixtures. The expected fractional values are shown in the third column of Table II. The SD,, value of the mixture components calculated from fractional values actually found are shown in the fourth column. These “found” values were calculated by dividing the SD70 value found in the mixture SDTo by the dose rate level value found when the compound was given alone, e.g., in the 3:l combination. The sulfadiazine found fractional value shown in Column 4 is 0.22 (0.033 MKD/0.15 MKD = 0.22). The expected to found ratios are shown in the last column and are obtained by dividing the expected fractional value in Column 3 by the corresponding found fractional value in Column 4. In the case of the 3:l ratio, the expected to found ratios are 2.27 and 15.63 for sulfadiazine and WR 158,122, respectively. The best combination may be observed by noting in Column 2 which MKD is smallest. Inspection of Table II, Column 2 reveals that combination to be 3:I. Other analyses are similarly conducted. A graphical representation of the values obtained in TabIe II is shown in Fig. 3. The use of this type diagram, introduced by Loewe and described by Gaddum
IN
MALARIA
99
THERAPY
AI 4 More Than
0
0.2
Addltlve
Acllon
0.4
Proportional WR 158,122
0.6
Amount
(Potanttati
08 of on SD,,
IO of
or WR 180,872
FIG. 3. Loewe diagram illustrating the degree of potentiation produced in effecting 70% suppression of parasitemia of P. berghi infected mice by mixing WR 158,122 or WR 180,872 and sulfadiazine for 3 day treatment. The positions of the combination points below the line of addition are evidence of potentiation.
(1959), is obtained by plotting the found fractional values in Table II, Column 4. The Fig. 3 diagram represents the combination of sulfadiazine and either WR 158,122 or WR 180,872. The x axis represents the proportional amount of sulfadiazine which effects an SDTOin the absence of any other drug. Similarly, the y axis represents the proportional amount of WR 158,I22 or WR 180,872 to produce an SD70 when it is administered alone. If a combination of sulfadiazine and either WR 158,122 or WR 180,872 were administered and the found fractional value from Table II, Column 4 were such that it fell on the hypotenuse the conclusion would be that the drugs in that particular combination were simply additive in their effects. Further, if an SDTo were produced by a drug combination that when plotted fell below the hypotenuse, the conclusion would be that the drug combination was more additive, i.e., potentiation existed. On the other hand, if when treating, presence of WR 158,122 or WR 180,872 necessitated the presence of an increased quantity of sulfadiazine to produce an SD,,; or conversely if the presence of sulfadiazine required increased quan-
100
KINNAMON,
AGER
TABLE Data
Analysis of Joint Antimalarial berg&i Infected Mice, Based
Action of WR upon Partitioning
AND
OHCHARD
III
158,122 and Wlb 180,872 of Mixtures in Terms
SDd
Drugs
Drug
observed (mg/k/day) Expected additive 1. WR 158,122 2. WR 180,872 3. 1 Part WR 158,122 1 Part WR 180,872
0.88 1.1 0.325 0.325
if
upon I’arGlemis of Plasmodium of Fractions of the SD90 fractions effecting Found
of mixtures an SD90 Expected/ found
0.5 0.5
0.369 0.295
1.355 1.695
0.5 0.5
0.318 0.127
1.572 3.937
0.5 0.5
0.373 0.075
1.340 6.667
0.3 0.0c
0.403 0.032
1.241 15.625
0.5 0.5
0.248 0.456
2.016 1.097
0.5 0.5
0.218 0.800
2.294 0.625
0.5 0.5
0.104 0.947
4.808 0.528
0.5 0.5
0.045 1.042
11.111 0.480
0.5 0.5
0.472 0.018
1.059 27.778
0.65 4. 2 Parts WI1 158,122 1 Part WR 180,872
0.28 0.14
0.42 5. 4 Parts WR 158,122 1 Part WR 180,872
0.328 0.082 0.41
6. 10 Parts WR 158,122 1 Part WR 180,872
0.3546 0.0355 0.30
7. WR 158,122 8. WR 180,872 9. 1 Part WR 158,122 2 Parts WR 180,872
1.1 1.2 0.273 0.547 0.82
10. 1 Part WR 158,122 4 Parts WR 180,872
0.24 0.96 1.2
11.
1 Part WR 158 122 10 Parts WR 186,872
0.114 1.136 1.35
12.
1 Part WR 158,122 25 Parts WR 180,872
0.05 1.25 1.3
13. 25 Parts WR 158,122 1 Part WR 180,872
0.519 0.021 0.54
0 SD,,
= That
dose causing
a 90%
suppression
of parasitemia.
tities of WR 158,122 or WR 180,872 to produce an SD,, the conclusion would be that the drug combination was less than addi-
tive, i.e., antagonism existed. When points from such less than additive combinations 1 are plotted on a diagram such as in Fig. 3 i
FOLIC
ACID
ANTAGONIST
POTENTIATION
IN
MALARIA
101
THERAPY
the points
appear above the hypotenuse. It may be noted from Fig. 3 that all points from the sulfadiazine-antifol combinations used in the present study fell well below the line of addition. Synergism:
WR 158,122-WR
180,872
Combinations of the 2,4-diaminothioquinazoline, WR 158,122 and its tetrahydro analog, WR 180,872, were found to be synergistic in all ratios where WR 180,872 made up no more than two-thirds of the drug-combination. The data are summarized in Table III and Fig. 4. The 2:1, 4:1, and 1O:l ratios were most effective; the 25:l ratio less effective but slightly more potentiating than the 1: 1 and 1:2 ratios. The 1:4, 1: 10, and 1:25 ratios were approximately additive in effect. DISCUSSION
In this paper we present evidence that subsequent to treatment of 2’. herghei infected mice with combined doses of different antifolates there is a greater than additive chemotherapeutic effect of the two drugs. Thus, the drugs 2,4-diamino-6- ( 2naphthyl-sulfonyl )-quinazoline ( WR 158,122) and its tetrahydro analog (WR 180,872) are presumed to be acting synergistically by affecting two different sites in the folic acid metabolic pathway, analogous to potent&ion observed when sulfonamides and DHF reductase inhibitors are combined. Further, both of these sites of action are believed to be at a location other than Point A (Fig. 1) for two reasons. First sulfadiazine acts synergistically with either WR 158,122 or WR 180,872. Second, the activity of sulfadiazine is reversed by PABA, whereas the activity of the other two drugs is unaffected by PABA. Since in the metabolic pathway (Fig. 1) there is the conversion from dihydrofolate to tetrahydrofolate, one may suspect on the basis of closer structural similarity that the quinazoline WR 158,122 is active at Point B, whereas the tetrahydroquinazoline WR 180,872 is likely to be more
Proportuxwt
Amount of
of
an
SDgO
WR 180,872
FIG. 4. Loewe diagram illustrating the degree of potentiation produced in effecting 90% suppression of parasitemia of P. berghi infected mice by mixing WR 158,122 and WR 180,872 for 3 day treatment. The positions of the combination points below the line of addition are evidence of potentiation.
active at Point C. It is possible that one or both of these compounds is partially active at both sites. It has been pointed out that cofactors required for cle nom methyl synthesis in P. berghei include tetrahydrofolic acid and that the use of antifolates as antimalarials may serve to block that reaction as well as other folic acid dependent reactions (Langer et al. 1969) . In mammalian metabolic systems it is known that folate coenzymes are involved as carriers in possibly all reactions involving the transfer of one-carbon units, viz, -CH:
