Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
The chemotherapy of rodent malaria, XXIV D. C. Warhurst, B. L. Robinson & W. Peters To cite this article: D. C. Warhurst, B. L. Robinson & W. Peters (1976) The chemotherapy of rodent malaria, XXIV, Annals of Tropical Medicine & Parasitology, 70:3, 253-258, DOI: 10.1080/00034983.1976.11687121 To link to this article: https://doi.org/10.1080/00034983.1976.11687121
Published online: 15 Mar 2016.
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Annals of Tropical Medicine and Parasitology, Vol. 70, No.3 (1976)
The chemotherapy of rodent malaria, XXIV The blood schizontocidal action of erythromycin upon Plasmodium berghei BY D. C. WARHURST, B. L. ROBINSON
AND
W. PETERS
Department of Parasitology, Liverpool School of Tropical Medicine Received 29 July 1975 In preliminary studies the macrolide antibiotic erythromycin was the only antibacterial antibiotic we tested that would inhibit at I 0- 4 M chloroquine-induced pigment clumping (CIPC) in Plasmodium berghei in vitro (Warhurst et al., 1972). Tests carried out in vivo in mice confirmed the blood schizontocidal activity of erythromycin and indicated that it potentiates the action of chloroquine upon chloroquine-resistant strains (Robinson and Warhurst, 1972). In this paper we compare the effectiveness of base and stearate, given by oral and subcutaneous routes, and studies of potentiation are reported in more detail. MATERIALS AND METHODS The '4-day test' (Peters, 1965) was carried out on strain N (Kl73) (chloroquine- and quinine-sensitive), strain NS (chloroquine-resistant and quinine-sensitive) (Peters et al., 1970) and strain RC (chloroquine- and quinine-resistant) (Peters, 1965) of P. berghei in Eperythrozoon-free, random bred, male albino mice (Tuck's TFW line). The inoculum was injected intravenously into the tail vein (day 0) and mice were then randomized into cages prior to drug treatment, which was carried out orally (po) or subcutaneously (sc). Erythromycin base and stearate were homogenized in saline with a small quantity of Tween 80. Different sides of the body were used where two drugs were to be injected subcutaneously into the same animal. ED 90 values for drugs and drug combinations were calculated as described previously (Peters, 1965) and results of potentiation experiments were illustrated graphically as type I isoboles (DeJongh, 1961 ). Source of Drugs Chloroquine diphosphate was a gift of Imperial Chemical Industries, Ltd. Erythromycin base was obtained from Sigma {London) Ltd., and erythromycin stearate was donated by Abbott Laboratories, Queenborough, Kent. RESULTS Erythrom.ycin Alone Comparisons were made between the activity of erythromycin base and stearate This work was financed in part by the Medical Research Council and by the U.S. Research and Development Command, Department of the Army, under contract DAJA27-74-C-1496, through the European Research Office. In respect of the latter, this paper is contribution no. 1376 from the Army Research Program on Malaria.
CHEMOTHERAPY OF RODENT MALARIA XXIV
254
administered po or sc to mice inoculated with RC strain, and sc to mice with N and NS strains. The results are presented in the Table. TABLE
Blood schizontocidal activity of erythromycin against P. berghei in mice. ED 90 (calculated in terms of mgfkg base) given sc or po as base or stearate, daily for four days Strain of P. berghei
-
0)
---
Route
Base
Stearate
RC
po sc
>1000 480
430 > 1000
l.XS
sc
500
> 1000
N
sc
500
-1000
3
~
0)
D
E
N strain
~ ci. 2 w
'
Q)
C"'
,
....
c: :l
..• . .. ...
........ ,0, ....
1
'e . . . . . .......
0 .... 0
... ....
'
.. .... , '"[] ....
•
.. ..... ....
....
..L:.
u
100
200
300
400
500
Erythromycin
E.o.
600 90
.. 700
800
(mgjkg)
Fig. 1. The action of erythromycin and chloroquine given alone or in various combinations against P. berghei N strain in the '4-day test'. ORDINATE. Daily dose of chloroquine (as base) n mg/kg reducing parasitaemia to 10% ofundosed control (ED 10 ). ABSCISSA. Daily dose of erythromycin base in mg/kg reducing parasitaemia to 10% of unclosed control (ED 10 ). (See further explanation in text).
WARHURST et al.
255
It is apparent from the Table that, given sc, erythromycin base is more effective than the stearate against all three strains. When given po against the RC strain, the base is less effective than the stearate. Co~nbined
Eft"ect of Erythro~nycin and Chloroquine In the '4-day test' various combinations of chloroquine diphosphate and erythromycin base were administered sc to groups of five mice infected with N, NS or RC strains. In some groups the chloroquine dose was held constant and the daily dose of erythromycin varied. In other groups the erythromycin dose was held constant and the chloroquine dose varied. ED 90 values for erythromycin and chloroquine respectively were calculated from the mean percentage of erythrocytes found infected on day 4 in the dosed groups (compared with sham-dosed control groups). In Figures I, 2 and 3 these ED 90 values are plotted as isoboles; erythromycin ED 90 values in the presence of fixed doses of chloroquine are represented by open squares, whilst chloroquine ED 90 values in the presence of fixed doses of erythromycin are represented as closed circles. In the case of N strain (Fig. I), the ED 90 values observed do not differ from those predicted (broken line) on the hypothesis that no potentiation occurs between chloroquine and erythromycin. For NS strain (Fig. 2) a weak degree of potentiation is indicated, since
7
-
---E
6 '
~
5
0
P- 4
'
'''
''
''
''
'
''
.
0 w
3
•
NS strain '
''
''
.. .'
' ' ...
400
'
'
....
500
', . e 600
700
Erythromycin E.o.90 (mg/ kg) FIK. 2. The action of erythromycin and chloroquine given alone or in various combinations against P. berghei NS strain. (Other details as for Fig. 1).
CHEMOTHERAPY OF RODENT MALARIA XXIV
256 '
m4oo
''
~
.............
''
C)
E
00.3oo d. w (I)
''
'·
''
RCstrain ''
' '·
'
''
200
''
''
''
r:::::
''
::J
'·
''
c-
' ''
o 100 '0
'
''
''
..s::.
u
200
100 Erythromycin
E.o.
300
90
(m
'
'''
',
400
gf kg)
Fig. 3. The action of erythromycin and chloroquine given alone or in various combinations against P. berghei RC strain. (Other details as for Fig. 1).
points fall below and to the left of the broken line. In RC strain (Fig. 3) there is clearly strong potentiation between the two drugs. In a further experiment it was found that the therapeutic effect of chloroquineerythromycin combinations upon RC strain was unaffected by feeding the mice additional para-aminobenzoic acid (10 mg/1) in their drinking water.
DISCUSSION In man, erythromycin base given orally is to some extent destroyed by gastric acid. The use of erythromycin stearate leads to higher intestinal drug concentrations and higher plasma levels (Weinstein, 1970). These factors would explain the superiority of the stearate when used orally against RC strain in mice. It seems that when given subcutaneously the base is better absorbed than the stearate. Erythromycin markedly potentiated the blood schizontocidal action of chloroquine against the RC strain of P. berghei. A dose of 300 mgfkg lowered the chloroquine ED 90 from an estimated (toxic) level of over 400 mg/kg to 1·25 mg/kg, similar to the ED 90 for the N strain. Potentiation in the NS strain was not so marked, but was still detectable.
WARHURST et al.
257
Erythromycin at 150 mg/kg reduced the chloroquine ED 90 from 6 mgfkg to 2·5 mg/kg. P. berghei RC and NS strains are markedly different in character. The former is avirulent, unpigmented and reticulocyte-dwelling, whilst the latter is relatively virulent, pigmented and inhabits normocytes and reticulocytes (Peters, 1970). Nevertheless, potentiation between erythromycin and chloroquine is shown in both chloroquine-resistant strains, and not in the chloroquine-sensitive N strain. It therefore appears that the potentiation between the two drugs is related to chloroquine-resistance. It will not be possible to discuss adequately the reasons for potentiation until the mode of action of erythromycin itself against malaria parasites is known. Interestingly, macrolide antibiotics are active against two other members of the sporozoan subphylum Apicomplexa (Levine, 1970), Toxoplasma (McCowen et al., 1953) and Eimeria (Panitz, 1974). The rationale for the studies on potentiation carried out here was the hypothesis of Howells et al. (1970), who suggested that malaria parasites might elude the effects of chloroquine by an increase in the citric acid cycle and general mitochondrial activity (see also Howells et al., 1972). The results reported here do not conflict with this idea. There is evidence from CIPC that erythromycin acts directly on the malaria parasite, but it is conceivable that the effects in vivo result from an action of the drug upon the host's intestinal flora and consequent changes in the nutritional state of the mouse. However, additional dietary p-aminobenzoic acid had no antagonistic effect, and it was shown by Kaddu et al. (1974) that the tetracycline antibiotic minocycline, which might equally be expected to affect intestinal flora, did not potentiate the action of chloroquine upon chloroquine-resistant P. berghei strains. Subsequent work on P. knowlesi also suggests that erythromycin has a direct antimalarial effect.
SUMMARY Erythromycin inhibits chloroquine-induced pigment clumping in Plasmodium berghei in vitro. The drug was therefore tested against infections of P. berghei in mice and was found to be active at non-toxic doses. Given orally, the stearate salt was more effective than the base, but subcutaneously the base was more effective than the stearate. Erythromycin potentiated the action of chloroquine against two chloroquine-resistant strains of rodent malaria, the mildly resistant NS, and the highly resistant RC strains of P. berghei, but not against the drug-sensitive N strain. ACKNOWLEDGEMENTS. We gratefully acknowledge the support from Dr. E. W. Witherspoon and Abbot Laboratories, Queenborough, Kent, towards these experiments.
REFERENCES DEjONGH, S. E. (1961). 'lsoboles' in Quantitative Methods in Pharmacology, ed. Dejonge, H., (Amsterdam). HowELLS, R. E., PETERS, W. & HoMEWOOD, C. A. (1972). 'Physiological adaptability of malaria parasites.' In Comparative Biochemistry of Parasites, ed. Van den Bossche, H. (New York & London: Academic Press). HowELLS, R. E., PETERS, W., HoMEWOOD, C. A. & WARHURST, D. C. (1970). 'Theory for the mechanism of chloroquine-resistance in rodent malaria.' Nature, London, 228, 625-628. KADDU, j. B., WARHURST, D. C. & PETERS, W. (1974). 'The chemotherapy of rodent malaria, XIX. The action of a tetracycline derivative, minocycline, on drug-resistant Plasmodium berghei.' Annals of Tropical Medicine and Parasitology, 68, 41-46. LEVINE, N.D. (1970). 'Taxonomy of the Sporozoa.' Journal of Parasitology, 56,208-209. McCoWEN, M. C., CALLENDER, M. E., LAwus,j. F. & BRANDT, M. C. (1953). 'The effects of erythromycin (IIotycin, Lilly) against certain parasitic organisms.' American Journal of Tropical Medicine and Hygiene, 2, 212-218.
258
CHEMOTHERAPY OF RODENT MALARIA XXIV
PANITz, E. (1974). 'Anticoccidial activity of rosamycin.' Journal of Parasitology, 60, 530-531. PETERS, W. (1965). 'Drug resistance in Plas77Wdium berghei, Vincke and Lips, 1948. I. Chloroquine resistance.' Experimental Parasitology, 16, 158-166. PETERS, W. (1970). Chemotherapy and Drug Resistance in Malaria. (London and New York: Academic Press). PETERS, W., BAFORT, J. & RAMKARAN, A. E. (1970). 'The chemotherapy of rodent malaria. XI. Cyclically transmitted chloroquine-resistant variants of the Keyberg 173 strain of Plasmodium berghei.' Annals of Tropical Medicine and Parasitology, 64, 41-52. RoBINSON, B. L. & WARHURST, D. C. (1972). 'Antimalarial activity of erythromycin.' Transactions of the Royal Society of Tropical Medicine and Hygiene (Laboratory Demonstration), 66, 525. WARHURST, D. C., HoMEWOOD, C. A., PETERS, W. & BAGGALEY, V. C. (1972). 'Pigment changes in PlUS77Wdium berghei as indicators of activity and mode of action of antimalarial drugs.' Proceedings of the Helminthological Society of Washington (Special Issue: Basic Research in Malaria), 39, 271-278. WEINSTEIN, L. (1970). 'Antibiotics' In The Pharmacological basis of Therapeutics. ed. Goodman, L. S., and Gilman, A. 4th Edition. (New York: Macmillan Press).
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
The chemotherapy of rodent malaria, XXIV D. C. Warhurst, B. L. Robinson & W. Peters To cite this article: D. C. Warhurst, B. L. Robinson & W. Peters (1976) The chemotherapy of rodent malaria, XXIV, Annals of Tropical Medicine & Parasitology, 70:3, 253-258, DOI: 10.1080/00034983.1976.11687121 To link to this article: https://doi.org/10.1080/00034983.1976.11687121
Published online: 15 Mar 2016.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 20 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19
Annals of Tropical Medicine and Parasitology, Vol. 70, No.3 (1976)
The chemotherapy of rodent malaria, XXIV The blood schizontocidal action of erythromycin upon Plasmodium berghei BY D. C. WARHURST, B. L. ROBINSON
AND
W. PETERS
Department of Parasitology, Liverpool School of Tropical Medicine Received 29 July 1975 In preliminary studies the macrolide antibiotic erythromycin was the only antibacterial antibiotic we tested that would inhibit at I 0- 4 M chloroquine-induced pigment clumping (CIPC) in Plasmodium berghei in vitro (Warhurst et al., 1972). Tests carried out in vivo in mice confirmed the blood schizontocidal activity of erythromycin and indicated that it potentiates the action of chloroquine upon chloroquine-resistant strains (Robinson and Warhurst, 1972). In this paper we compare the effectiveness of base and stearate, given by oral and subcutaneous routes, and studies of potentiation are reported in more detail. MATERIALS AND METHODS The '4-day test' (Peters, 1965) was carried out on strain N (Kl73) (chloroquine- and quinine-sensitive), strain NS (chloroquine-resistant and quinine-sensitive) (Peters et al., 1970) and strain RC (chloroquine- and quinine-resistant) (Peters, 1965) of P. berghei in Eperythrozoon-free, random bred, male albino mice (Tuck's TFW line). The inoculum was injected intravenously into the tail vein (day 0) and mice were then randomized into cages prior to drug treatment, which was carried out orally (po) or subcutaneously (sc). Erythromycin base and stearate were homogenized in saline with a small quantity of Tween 80. Different sides of the body were used where two drugs were to be injected subcutaneously into the same animal. ED 90 values for drugs and drug combinations were calculated as described previously (Peters, 1965) and results of potentiation experiments were illustrated graphically as type I isoboles (DeJongh, 1961 ). Source of Drugs Chloroquine diphosphate was a gift of Imperial Chemical Industries, Ltd. Erythromycin base was obtained from Sigma {London) Ltd., and erythromycin stearate was donated by Abbott Laboratories, Queenborough, Kent. RESULTS Erythrom.ycin Alone Comparisons were made between the activity of erythromycin base and stearate This work was financed in part by the Medical Research Council and by the U.S. Research and Development Command, Department of the Army, under contract DAJA27-74-C-1496, through the European Research Office. In respect of the latter, this paper is contribution no. 1376 from the Army Research Program on Malaria.
CHEMOTHERAPY OF RODENT MALARIA XXIV
254
administered po or sc to mice inoculated with RC strain, and sc to mice with N and NS strains. The results are presented in the Table. TABLE
Blood schizontocidal activity of erythromycin against P. berghei in mice. ED 90 (calculated in terms of mgfkg base) given sc or po as base or stearate, daily for four days Strain of P. berghei
-
0)
---
Route
Base
Stearate
RC
po sc
>1000 480
430 > 1000
l.XS
sc
500
> 1000
N
sc
500
-1000
3
~
0)
D
E
N strain
~ ci. 2 w
'
Q)
C"'
,
....
c: :l
..• . .. ...
........ ,0, ....
1
'e . . . . . .......
0 .... 0
... ....
'
.. .... , '"[] ....
•
.. ..... ....
....
..L:.
u
100
200
300
400
500
Erythromycin
E.o.
600 90
.. 700
800
(mgjkg)
Fig. 1. The action of erythromycin and chloroquine given alone or in various combinations against P. berghei N strain in the '4-day test'. ORDINATE. Daily dose of chloroquine (as base) n mg/kg reducing parasitaemia to 10% ofundosed control (ED 10 ). ABSCISSA. Daily dose of erythromycin base in mg/kg reducing parasitaemia to 10% of unclosed control (ED 10 ). (See further explanation in text).
WARHURST et al.
255
It is apparent from the Table that, given sc, erythromycin base is more effective than the stearate against all three strains. When given po against the RC strain, the base is less effective than the stearate. Co~nbined
Eft"ect of Erythro~nycin and Chloroquine In the '4-day test' various combinations of chloroquine diphosphate and erythromycin base were administered sc to groups of five mice infected with N, NS or RC strains. In some groups the chloroquine dose was held constant and the daily dose of erythromycin varied. In other groups the erythromycin dose was held constant and the chloroquine dose varied. ED 90 values for erythromycin and chloroquine respectively were calculated from the mean percentage of erythrocytes found infected on day 4 in the dosed groups (compared with sham-dosed control groups). In Figures I, 2 and 3 these ED 90 values are plotted as isoboles; erythromycin ED 90 values in the presence of fixed doses of chloroquine are represented by open squares, whilst chloroquine ED 90 values in the presence of fixed doses of erythromycin are represented as closed circles. In the case of N strain (Fig. I), the ED 90 values observed do not differ from those predicted (broken line) on the hypothesis that no potentiation occurs between chloroquine and erythromycin. For NS strain (Fig. 2) a weak degree of potentiation is indicated, since
7
-
---E
6 '
~
5
0
P- 4
'
'''
''
''
''
'
''
.
0 w
3
•
NS strain '
''
''
.. .'
' ' ...
400
'
'
....
500
', . e 600
700
Erythromycin E.o.90 (mg/ kg) FIK. 2. The action of erythromycin and chloroquine given alone or in various combinations against P. berghei NS strain. (Other details as for Fig. 1).
CHEMOTHERAPY OF RODENT MALARIA XXIV
256 '
m4oo
''
~
.............
''
C)
E
00.3oo d. w (I)
''
'·
''
RCstrain ''
' '·
'
''
200
''
''
''
r:::::
''
::J
'·
''
c-
' ''
o 100 '0
'
''
''
..s::.
u
200
100 Erythromycin
E.o.
300
90
(m
'
'''
',
400
gf kg)
Fig. 3. The action of erythromycin and chloroquine given alone or in various combinations against P. berghei RC strain. (Other details as for Fig. 1).
points fall below and to the left of the broken line. In RC strain (Fig. 3) there is clearly strong potentiation between the two drugs. In a further experiment it was found that the therapeutic effect of chloroquineerythromycin combinations upon RC strain was unaffected by feeding the mice additional para-aminobenzoic acid (10 mg/1) in their drinking water.
DISCUSSION In man, erythromycin base given orally is to some extent destroyed by gastric acid. The use of erythromycin stearate leads to higher intestinal drug concentrations and higher plasma levels (Weinstein, 1970). These factors would explain the superiority of the stearate when used orally against RC strain in mice. It seems that when given subcutaneously the base is better absorbed than the stearate. Erythromycin markedly potentiated the blood schizontocidal action of chloroquine against the RC strain of P. berghei. A dose of 300 mgfkg lowered the chloroquine ED 90 from an estimated (toxic) level of over 400 mg/kg to 1·25 mg/kg, similar to the ED 90 for the N strain. Potentiation in the NS strain was not so marked, but was still detectable.
WARHURST et al.
257
Erythromycin at 150 mg/kg reduced the chloroquine ED 90 from 6 mgfkg to 2·5 mg/kg. P. berghei RC and NS strains are markedly different in character. The former is avirulent, unpigmented and reticulocyte-dwelling, whilst the latter is relatively virulent, pigmented and inhabits normocytes and reticulocytes (Peters, 1970). Nevertheless, potentiation between erythromycin and chloroquine is shown in both chloroquine-resistant strains, and not in the chloroquine-sensitive N strain. It therefore appears that the potentiation between the two drugs is related to chloroquine-resistance. It will not be possible to discuss adequately the reasons for potentiation until the mode of action of erythromycin itself against malaria parasites is known. Interestingly, macrolide antibiotics are active against two other members of the sporozoan subphylum Apicomplexa (Levine, 1970), Toxoplasma (McCowen et al., 1953) and Eimeria (Panitz, 1974). The rationale for the studies on potentiation carried out here was the hypothesis of Howells et al. (1970), who suggested that malaria parasites might elude the effects of chloroquine by an increase in the citric acid cycle and general mitochondrial activity (see also Howells et al., 1972). The results reported here do not conflict with this idea. There is evidence from CIPC that erythromycin acts directly on the malaria parasite, but it is conceivable that the effects in vivo result from an action of the drug upon the host's intestinal flora and consequent changes in the nutritional state of the mouse. However, additional dietary p-aminobenzoic acid had no antagonistic effect, and it was shown by Kaddu et al. (1974) that the tetracycline antibiotic minocycline, which might equally be expected to affect intestinal flora, did not potentiate the action of chloroquine upon chloroquine-resistant P. berghei strains. Subsequent work on P. knowlesi also suggests that erythromycin has a direct antimalarial effect.
SUMMARY Erythromycin inhibits chloroquine-induced pigment clumping in Plasmodium berghei in vitro. The drug was therefore tested against infections of P. berghei in mice and was found to be active at non-toxic doses. Given orally, the stearate salt was more effective than the base, but subcutaneously the base was more effective than the stearate. Erythromycin potentiated the action of chloroquine against two chloroquine-resistant strains of rodent malaria, the mildly resistant NS, and the highly resistant RC strains of P. berghei, but not against the drug-sensitive N strain. ACKNOWLEDGEMENTS. We gratefully acknowledge the support from Dr. E. W. Witherspoon and Abbot Laboratories, Queenborough, Kent, towards these experiments.
REFERENCES DEjONGH, S. E. (1961). 'lsoboles' in Quantitative Methods in Pharmacology, ed. Dejonge, H., (Amsterdam). HowELLS, R. E., PETERS, W. & HoMEWOOD, C. A. (1972). 'Physiological adaptability of malaria parasites.' In Comparative Biochemistry of Parasites, ed. Van den Bossche, H. (New York & London: Academic Press). HowELLS, R. E., PETERS, W., HoMEWOOD, C. A. & WARHURST, D. C. (1970). 'Theory for the mechanism of chloroquine-resistance in rodent malaria.' Nature, London, 228, 625-628. KADDU, j. B., WARHURST, D. C. & PETERS, W. (1974). 'The chemotherapy of rodent malaria, XIX. The action of a tetracycline derivative, minocycline, on drug-resistant Plasmodium berghei.' Annals of Tropical Medicine and Parasitology, 68, 41-46. LEVINE, N.D. (1970). 'Taxonomy of the Sporozoa.' Journal of Parasitology, 56,208-209. McCoWEN, M. C., CALLENDER, M. E., LAwus,j. F. & BRANDT, M. C. (1953). 'The effects of erythromycin (IIotycin, Lilly) against certain parasitic organisms.' American Journal of Tropical Medicine and Hygiene, 2, 212-218.
258
CHEMOTHERAPY OF RODENT MALARIA XXIV
PANITz, E. (1974). 'Anticoccidial activity of rosamycin.' Journal of Parasitology, 60, 530-531. PETERS, W. (1965). 'Drug resistance in Plas77Wdium berghei, Vincke and Lips, 1948. I. Chloroquine resistance.' Experimental Parasitology, 16, 158-166. PETERS, W. (1970). Chemotherapy and Drug Resistance in Malaria. (London and New York: Academic Press). PETERS, W., BAFORT, J. & RAMKARAN, A. E. (1970). 'The chemotherapy of rodent malaria. XI. Cyclically transmitted chloroquine-resistant variants of the Keyberg 173 strain of Plasmodium berghei.' Annals of Tropical Medicine and Parasitology, 64, 41-52. RoBINSON, B. L. & WARHURST, D. C. (1972). 'Antimalarial activity of erythromycin.' Transactions of the Royal Society of Tropical Medicine and Hygiene (Laboratory Demonstration), 66, 525. WARHURST, D. C., HoMEWOOD, C. A., PETERS, W. & BAGGALEY, V. C. (1972). 'Pigment changes in PlUS77Wdium berghei as indicators of activity and mode of action of antimalarial drugs.' Proceedings of the Helminthological Society of Washington (Special Issue: Basic Research in Malaria), 39, 271-278. WEINSTEIN, L. (1970). 'Antibiotics' In The Pharmacological basis of Therapeutics. ed. Goodman, L. S., and Gilman, A. 4th Edition. (New York: Macmillan Press).
