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High susceptibility of five axenic Entamoeba histolytica strains to gossypol
522
Tms~crto~s OPTHBROYAL. SOCIE~OPTROPICAL MEDICINE ANDHYGIENE(1989) 83, 522-524
High susceptibility
of five axenic Entamoeba
histolyfica
strains to gossypol
Ma. Teresa Gonzalez-Garza, Renito D. Mata-Chrdenas and Salvador Said-Femhdez Ditisih de St&g&a Celular, Unidad de InvestigaGn Biomkdica &I Noreste, Ins&to Mexican0 de1SeguroSocial, Apartado Postal, 020-E 64720 Monterrey, Nuevo Le&, Mkko Abstract The antiamoebic potency of gossypol was tested against 5 axe& Entanweba hiswlytica strains, in logarithmic phase growth in PEHPS medium. All of the strains were moderately susceptible to this polyphenolic drug. The 50% inhibitory concentration (I&) was of the same order of magnitude in all strains: O-015 pM (for strain HM-1) to O-067 w (for HM-38). The difference between the I& of HM-1 and the remaining 4 straius (HM-38, HK-9, HM-2 and HM-3) was sign&ant, although it was greater between I-IM-1 and HM-38 (P
38:IMSS (cited hereafter as HM-1, HM-2, HM-3 and HM-38, respectively) and HK-9 were grown in 16x 125 mm screw-capped tubes with 11 ml of PEHPS medium (SAID-FERNANDEZet al., 1988), incubated at 36*5“C and subcultivated every 4 d. Drug potmcy tests. These were performed as described by GONZALEZ-GARZA& SAID-FERNANDEZ (1988). Briefly, different concentrations of gossypol between O*OlO pM and O-200 w were added in triplicate to cultures with 1000amoebaeper ml, at the beginning of the logarithmic growth phase. Treated cultures were incubated for 72 h at 36*5”C, and cooled in ice water for 10 min. The number of trophozoites per ml was determined with a haemocytometer and the percentageof growth inhibition with respect to the untreated controls was determined. The 50% (I(&) inhibitory concentration was calculated by probit analysis (FINNEY, 1977). Statistical analysis. Student’s t test was used to determine the significance of differences between the I&, of gossypol with the 5 E. hiswlytica strains. Slope and elevation of the amoebic dose-responsecurves were compared by analysis of covariance (ZAR, 1974). Chemicals.Gossypol acetic acid was purchased from Sigma (St Louis, Missouri, USA); other salts were from J. T. Baker de M&&o (Xalostoc, M&co); all of them were reagent grade. Sterile bovine serum and PEHPS medium were prepared in our laboratory (SAID-FERNANDEZet al., 1988). Results The Figure shows the probit dose-responsecurve obtained with each amoebalstrain, which corresponds
& SAID-FERNANDEZ, 1988), 3 of the more widely
used drugs in antiamoebic chemotherapy (BIAGI, 1981). We have now extended the in vitro studies by examining the effect of gossypol on 4 axenic strains, in addition to HM-1, and we found that the inhibitory p$yyefmhis polyphenolic compound is similar with log OF DRUG CONCENTRATION (#+I)
Materials and Methods Amoebae. Trophozoites of E. hiswbtica strains HM-l:IMSS, HM-2:IMSS, HM-3:IMSS, HM-
F&m.
Dose-response linear relationship of gossypol against five
hisro&ica suains: HM-1 (A), HM-2 (A), HM-3 (0), HM-38(0) andHK-9 (0). J&h point reprrsentstheaverageof three
Enramwba
d&rent
experiments done in triplicate.
523 Table 1. Zh v&u antiamoebic activity of gossypol against five Eatamoeba liistolytica strains Confidence limits
Strain
Go8
HM-1 HK-9
0.015 0.030 O-038 0.049 0.067
iE:Z HM-38
0~010-0~021 0.022-0.038 O-027-0*049 0.031-0*067 0*045-0*089
“50% inhibitory concentration in micromoles. Table 2. In vitro antiamoebic drugs Emetine
potency
of several
0.082-1.6
NE.+L, 1978, 1983; tiE?-,E et a[., 1987; GONZALEZ-GNUA & SAIDFEW&DEL 1988.
Dchydroemetine
0.125-1.0
CHINTANA et al., 1986.
Meuonidazole
0.027-12.5
NWL, 1978; BHUTANI et al., 1987; CHATTERJEE et al., 1987; GONZALEZ-GARZA & SAIDFERNANDEZ, 1988.
Tinidazole
0.032-0.125
CHINTANA et al., 1986; NEAL, 1978.
Omidazole
0.032-0.125
CHINTANA et al., 1986; NEAL, 1978.
0.88
Diloxanide Diloxanide
furnate
Diiodohydroxyquinoline
GOSSYpOl
NUIL, 1978.
1.50-1.95
CHAITERJEE et ol., 1987; NEAL, 1978.
3.6-8.4
NEAL, 1978, 1983; GONZALEZ-GARZA & SAIDFERNANDEZ, 1988.
0+08-0~034
This paper.
‘Data obtainedfrom tests performedwith groupsof different E. histolyticaaxenic strains.
to a straight line in all cases. Covariance analysis showed no difference either in the regression coefficient or in slope elevation among all-these curves. The ICso and confidence limits determined from the data shown in the Figure are given in Table 1. All the I&o values determined were of the same order of magnitude between O-015 VM (Strain HM-1) and 0.067 pd (HM-38). Statistical analysis of the ICso values showed significant differences between that of HM- 1 and those of the remaining strains, even though the difference between HM-1 and HM-38 was greater (P
In a previous paper, we reported that gossypol has a potent antiamoebic effect in vitro upon E. hisrolyricu strain HM-1 (GONZALEZ-GARZA& SAID-FERNANDEZ, 1988). In the present work we extended our study by determining the inhibitory effect of gossypol on 4 other axenic E. histolyticu strains; all of them also were very susceptible to gossypol, with a narrow range of ICW values (0*010-O-070 uM), which included that df HM-I. &eater variat&ns’& ICso have been obtained by testing groups of several axenic
strains with other antiamoebic drugs, such as emetine (O-145-3-32 PM) (NEAL, 1978, 1983; KEENE et al., 1987), dehydroemetine (0.261-2-08 w) (CHINTANA et al., 1986), metronidazole (O-058-73*03 w) (NEAL, 1978; BHUTANI et al., 1987; CHATTERJEEet al., 1987), omidazole (0.145-O-569 pM) (NEAL, 1978; CHINTANAet aZ., 1986), tinidazole (0*129-O-505 PM) (NEAL, 1978; CHINTANAet al., 1986), and diiodohydroxyquinohne (7.521-30-713 PM) (NEAL, 1978, 1983; GONZALEZ-GARZA & SAID-FERNANDEZ,1988). The inhibitory potency in tirro of gossypol against strain HM-1 is noticeably greater than that of emetine, metronidazole, id- diiodohydroxyquinoline. 11. 39 and 980 times more active. resnectivelv (Go~z,~LEz-GARZA,& SAID-FERNAND&, i988). if the results reported in this and other papers are representative, it appears that the amoebicidal effect of gossypol on E. histolyticu is more potent, not only than the effect of the 3 compounds we tested, but also than the effect of several other antiamoebic drugs (Table 2). The pharmacological and toxicological properties of gossypol are currently being studied, because of its antifertilizing effect on humans. A group of healthy male volunteers received gossypol during long periods of time (10 years), and the great majority of them showed no undesirable clinical effects at the end of treatment; only a few individuals suffered hypokalaemia (XV, et al., 1988; LIU et al., 1988). In humans the an&fertilizin~ do& is 20 m&d foi 2 months, followed bv a maintenance doseof 50 melweek (Xu er al., 1988).aMetabolicstudies in humans~emplo$& a single dose of 20 mg, showed that gossypol reaches serum levels of 0.9% mg/litre 4-6 h after oral administration (WANG et al., 1985). In rats, after a single 50 mg dose, the drug was concentrated mainlv in Ihe organs more frequetitly colonized by amoebae in humans-liver (5*3%‘,and colon (56%-d it was eliminated in bil; via ‘the faeces (As&-DORIA & DIECKERT, 1974, 1975; JENSEN et al., 1982). From
these results we calculate that gossypol reacheslevels in serum, liver and colon which are much higher than the ICso determined in vitro with all the tested strains
(see Table 1).
All the above mentioned characteristics strongly support the idea that gossypol could be a good
alternative for antiamoebic medication, if it is equally potent in vivo against invasive trophozoites. Acknowledgements We thank Juan Antonio Luna de la Rosafor graphic work.
References
Abou-Donia, M. B. & Dieckert, J. W. (1974). Urinary and biliary excretion of “C-gossypol. Journal of Numition, 104, 754-760.
Abou-Donia, M. B. & Dieckert, J. W. (1975). Metabolic fate of gossypol: tbe metabolism of [14C]-gossypol in swine. Toxicology and Applied Pharmacology, 31, 32-46. Aitken, B. J. (1983). New techniques in conrraception: gossypol, vaccines and G&b analogues. In: Developments in Human Reproduction and their Eugenic Ethical Zm~lications, Carter, C. Q. (editor). New York: &ademic press, pp. 1-4. Bia$!?(1981). Amebiasis. Antiparasitic Chemothera~, 30, Bhutani, K. K., Shanna, G. L. & Ali,, M. (1987). Plant based anriamoebic drugs. Parr I. Anuamoebic activity of phenanrhroindolizine alkaloids; common structural de-
524
terminants of activity with emetine. Planta Medica, 53, 532-536. Blanco, A., Aoki, A., Montamat, E. E. & Rovai, L. E. (1983). Effect of gossypol upon motility and ultrastructure of Typanosoma cruzi 3ournal of Protomology, 30, 648-651. Campbell., W. C. (1986). The chemotherapy of parasitic infections. Journal of Parasiwlogy, 72, 45-61. Chang, M. C., Zhiping, Gu. & Saksena, S. K. (1980). Effects of gossypol on male rats, hamsters and rabbits. Contraception, 21, 461-469. Chatterjee, D. K., Iyer, N. & Ganguli, B. N. (1987). Antiamoebic activity of chonemorphine, a steroidal alkaloid, in experimental models. Parasiwlogy Research, 74. 30-33. Chintana, T., Sucharit, P., Mabakittikun, V., Siripanth, C. & Suphadtanaphongs,W. (1986). In vitro studies on the sensitivitv of local Entamoeba hiswlvtia to anti-amoebic drugs. S&&east Asian 3oumal of &pica1 Medicine and Public Health, 17, 591-594. Dorsett, P. H. & Kerstine, E. (1975). Antiviral activity of gossypol and apogossypol. 3oumal of Pharmaceutical Sciences, 64, 1073-1075. Finney, D. L. (1977). Probit Analysis. New York: Cambridge University Press. Gonzalez-Garza, M. T. & Said-FemBndez, S. (1988). Entamoeba hisw&ica: potent in vitro antiamoebic effect of gossypol. Experimental Parasiwlogy, 66, 253-255. Jensen, D. R., Sorensen, J. M. & Bosek, S. A. (1982). Deposition pattern of antifertility agent, gossypol, in selected organs of male rats. Toxicology, 24, 65-72. Keene, A. T., Phillipson, J. D., Warhurst, D. C., Koch, M. & Seguin, E. (1987). In tifro amoebicidal testing of natural products. Part 2. Alkaloids related to emetine. Planta Medica, 4, 201-206. Liu, G.-z., Ch’iu-Hinton, K., Cao, J., Zhu, C. & Li, B. (1988). Effects of K salts or potassium blocker on gossypol-related hypokalemia. Cuntracepfiun, 37, 11l_.III. Montamat, E. E., Burgos, C., Gerez de Burgos, N. M., Rovai. L. E. & Blanco. A. (1982). Inhibitorv action of gossy& on enzymes &d grbwth’of Trypanokma cnui. Science, 218, 288-289. Neal? R. A. (1978). Antiamoebic activity of drugs given smgly and in combination against axenically grown Entmweba hiswlvtica. Archives de Znvestipaci& MLdica (Mexico), 9, (subplement), 387-392. -
Neal, R. A. (1983). Experimental amoebiasis 9d developTp;:9;f anti-amoebic compounds. Paraslwlogy, 86, Radloff, R. ‘J., Deck, L M., Royer, R. E. & Vander Jagt, D. L. (1986). Anti&al activities of gossypol and its derivates against herpes simplex virus type II. Phannacological Research Communications,18, 1063-1073. Rikihisa, Y. & Lin, Y. C. (1986). Taenia taeniaefmis: inhibition of metacestode development in the rat by gossypol. Experimental Parasiwloa, 61, 127-133. Royer, R. E., Deck, L. M., Campos, N. M., Hunsaker, L. A. & Vander Jagt, D. L. (1986). Biologically active derivates of gossypol: synthesisand antimalarial activities of peri-acylated gossylic nitriles. 3oumal of Medicinal C&ma&y, 29, 1799-1801. Said-Femhdez, S., Vargas-Villareal, J., Castro-Garza, J., Mata-Cardenas, B. D., Navarro-Marmolejo, L., LozanoGarza, G. & Martinez-Rodriguez, H. (1988). PEHPS medium: an alternative for axenic cultivation of EnTransact@ of the tamoeba hiswlytica and ,E. invad?. f4~jLociery
of Troprcal Medmne and Hygaene, 82,
Van der Jab, D. L., Heidrich, J. E., Royer, R. E. & Hunsaker, L. (1982). Antimalarial activity of gossypol and methylglyoxal-bis-guanylhydrazone. Federation Proceedings, 41, 1428. Wang, M. Q., Wu, E. F. & Yu, Y. W. (1985). Highperformance liquid chromatography with electrochemical detection of gossypol in human plasma. 3o~nal of Chromarography and Biochemical Applications, 343, 387-
3%. Wickmann, K., Vaheri, A. & Luukkainen, T. (1982). Inhibition of herpes simplex virus type 2 infection in human epithelial cells by gossypol, a potent spermicidal and contraceptive agent. American Journal of Obstetrics and Gynecology, 142, 593-594. Xu, D., Cai, W.-j., Zhu,B.-j., Dong, C.-j., Zheng, Z.-c. 81 Gao?Z.-q. (1988). Clinical safety of long-term administranon of gossypol in 32 cases. Contraception, 37, 129-135. Zar, J. H. (1974). Btistatistical Analjrsis. Englewood Cliffs, New Jersey: Prentice-Hall, pp. 228-235.
Received 24 October 1988; accepted Januuy 1989
for publication
25
Tms~crto~s OPTHBROYAL. SOCIE~OPTROPICAL MEDICINE ANDHYGIENE(1989) 83, 522-524
High susceptibility
of five axenic Entamoeba
histolyfica
strains to gossypol
Ma. Teresa Gonzalez-Garza, Renito D. Mata-Chrdenas and Salvador Said-Femhdez Ditisih de St&g&a Celular, Unidad de InvestigaGn Biomkdica &I Noreste, Ins&to Mexican0 de1SeguroSocial, Apartado Postal, 020-E 64720 Monterrey, Nuevo Le&, Mkko Abstract The antiamoebic potency of gossypol was tested against 5 axe& Entanweba hiswlytica strains, in logarithmic phase growth in PEHPS medium. All of the strains were moderately susceptible to this polyphenolic drug. The 50% inhibitory concentration (I&) was of the same order of magnitude in all strains: O-015 pM (for strain HM-1) to O-067 w (for HM-38). The difference between the I& of HM-1 and the remaining 4 straius (HM-38, HK-9, HM-2 and HM-3) was sign&ant, although it was greater between I-IM-1 and HM-38 (P
38:IMSS (cited hereafter as HM-1, HM-2, HM-3 and HM-38, respectively) and HK-9 were grown in 16x 125 mm screw-capped tubes with 11 ml of PEHPS medium (SAID-FERNANDEZet al., 1988), incubated at 36*5“C and subcultivated every 4 d. Drug potmcy tests. These were performed as described by GONZALEZ-GARZA& SAID-FERNANDEZ (1988). Briefly, different concentrations of gossypol between O*OlO pM and O-200 w were added in triplicate to cultures with 1000amoebaeper ml, at the beginning of the logarithmic growth phase. Treated cultures were incubated for 72 h at 36*5”C, and cooled in ice water for 10 min. The number of trophozoites per ml was determined with a haemocytometer and the percentageof growth inhibition with respect to the untreated controls was determined. The 50% (I(&) inhibitory concentration was calculated by probit analysis (FINNEY, 1977). Statistical analysis. Student’s t test was used to determine the significance of differences between the I&, of gossypol with the 5 E. hiswlytica strains. Slope and elevation of the amoebic dose-responsecurves were compared by analysis of covariance (ZAR, 1974). Chemicals.Gossypol acetic acid was purchased from Sigma (St Louis, Missouri, USA); other salts were from J. T. Baker de M&&o (Xalostoc, M&co); all of them were reagent grade. Sterile bovine serum and PEHPS medium were prepared in our laboratory (SAID-FERNANDEZet al., 1988). Results The Figure shows the probit dose-responsecurve obtained with each amoebalstrain, which corresponds
& SAID-FERNANDEZ, 1988), 3 of the more widely
used drugs in antiamoebic chemotherapy (BIAGI, 1981). We have now extended the in vitro studies by examining the effect of gossypol on 4 axenic strains, in addition to HM-1, and we found that the inhibitory p$yyefmhis polyphenolic compound is similar with log OF DRUG CONCENTRATION (#+I)
Materials and Methods Amoebae. Trophozoites of E. hiswbtica strains HM-l:IMSS, HM-2:IMSS, HM-3:IMSS, HM-
F&m.
Dose-response linear relationship of gossypol against five
hisro&ica suains: HM-1 (A), HM-2 (A), HM-3 (0), HM-38(0) andHK-9 (0). J&h point reprrsentstheaverageof three
Enramwba
d&rent
experiments done in triplicate.
523 Table 1. Zh v&u antiamoebic activity of gossypol against five Eatamoeba liistolytica strains Confidence limits
Strain
Go8
HM-1 HK-9
0.015 0.030 O-038 0.049 0.067
iE:Z HM-38
0~010-0~021 0.022-0.038 O-027-0*049 0.031-0*067 0*045-0*089
“50% inhibitory concentration in micromoles. Table 2. In vitro antiamoebic drugs Emetine
potency
of several
0.082-1.6
NE.+L, 1978, 1983; tiE?-,E et a[., 1987; GONZALEZ-GNUA & SAIDFEW&DEL 1988.
Dchydroemetine
0.125-1.0
CHINTANA et al., 1986.
Meuonidazole
0.027-12.5
NWL, 1978; BHUTANI et al., 1987; CHATTERJEE et al., 1987; GONZALEZ-GARZA & SAIDFERNANDEZ, 1988.
Tinidazole
0.032-0.125
CHINTANA et al., 1986; NEAL, 1978.
Omidazole
0.032-0.125
CHINTANA et al., 1986; NEAL, 1978.
0.88
Diloxanide Diloxanide
furnate
Diiodohydroxyquinoline
GOSSYpOl
NUIL, 1978.
1.50-1.95
CHAITERJEE et ol., 1987; NEAL, 1978.
3.6-8.4
NEAL, 1978, 1983; GONZALEZ-GARZA & SAIDFERNANDEZ, 1988.
0+08-0~034
This paper.
‘Data obtainedfrom tests performedwith groupsof different E. histolyticaaxenic strains.
to a straight line in all cases. Covariance analysis showed no difference either in the regression coefficient or in slope elevation among all-these curves. The ICso and confidence limits determined from the data shown in the Figure are given in Table 1. All the I&o values determined were of the same order of magnitude between O-015 VM (Strain HM-1) and 0.067 pd (HM-38). Statistical analysis of the ICso values showed significant differences between that of HM- 1 and those of the remaining strains, even though the difference between HM-1 and HM-38 was greater (P
In a previous paper, we reported that gossypol has a potent antiamoebic effect in vitro upon E. hisrolyricu strain HM-1 (GONZALEZ-GARZA& SAID-FERNANDEZ, 1988). In the present work we extended our study by determining the inhibitory effect of gossypol on 4 other axenic E. histolyticu strains; all of them also were very susceptible to gossypol, with a narrow range of ICW values (0*010-O-070 uM), which included that df HM-I. &eater variat&ns’& ICso have been obtained by testing groups of several axenic
strains with other antiamoebic drugs, such as emetine (O-145-3-32 PM) (NEAL, 1978, 1983; KEENE et al., 1987), dehydroemetine (0.261-2-08 w) (CHINTANA et al., 1986), metronidazole (O-058-73*03 w) (NEAL, 1978; BHUTANI et al., 1987; CHATTERJEEet al., 1987), omidazole (0.145-O-569 pM) (NEAL, 1978; CHINTANAet aZ., 1986), tinidazole (0*129-O-505 PM) (NEAL, 1978; CHINTANAet al., 1986), and diiodohydroxyquinohne (7.521-30-713 PM) (NEAL, 1978, 1983; GONZALEZ-GARZA & SAID-FERNANDEZ,1988). The inhibitory potency in tirro of gossypol against strain HM-1 is noticeably greater than that of emetine, metronidazole, id- diiodohydroxyquinoline. 11. 39 and 980 times more active. resnectivelv (Go~z,~LEz-GARZA,& SAID-FERNAND&, i988). if the results reported in this and other papers are representative, it appears that the amoebicidal effect of gossypol on E. histolyticu is more potent, not only than the effect of the 3 compounds we tested, but also than the effect of several other antiamoebic drugs (Table 2). The pharmacological and toxicological properties of gossypol are currently being studied, because of its antifertilizing effect on humans. A group of healthy male volunteers received gossypol during long periods of time (10 years), and the great majority of them showed no undesirable clinical effects at the end of treatment; only a few individuals suffered hypokalaemia (XV, et al., 1988; LIU et al., 1988). In humans the an&fertilizin~ do& is 20 m&d foi 2 months, followed bv a maintenance doseof 50 melweek (Xu er al., 1988).aMetabolicstudies in humans~emplo$& a single dose of 20 mg, showed that gossypol reaches serum levels of 0.9% mg/litre 4-6 h after oral administration (WANG et al., 1985). In rats, after a single 50 mg dose, the drug was concentrated mainlv in Ihe organs more frequetitly colonized by amoebae in humans-liver (5*3%‘,and colon (56%-d it was eliminated in bil; via ‘the faeces (As&-DORIA & DIECKERT, 1974, 1975; JENSEN et al., 1982). From
these results we calculate that gossypol reacheslevels in serum, liver and colon which are much higher than the ICso determined in vitro with all the tested strains
(see Table 1).
All the above mentioned characteristics strongly support the idea that gossypol could be a good
alternative for antiamoebic medication, if it is equally potent in vivo against invasive trophozoites. Acknowledgements We thank Juan Antonio Luna de la Rosafor graphic work.
References
Abou-Donia, M. B. & Dieckert, J. W. (1974). Urinary and biliary excretion of “C-gossypol. Journal of Numition, 104, 754-760.
Abou-Donia, M. B. & Dieckert, J. W. (1975). Metabolic fate of gossypol: tbe metabolism of [14C]-gossypol in swine. Toxicology and Applied Pharmacology, 31, 32-46. Aitken, B. J. (1983). New techniques in conrraception: gossypol, vaccines and G&b analogues. In: Developments in Human Reproduction and their Eugenic Ethical Zm~lications, Carter, C. Q. (editor). New York: &ademic press, pp. 1-4. Bia$!?(1981). Amebiasis. Antiparasitic Chemothera~, 30, Bhutani, K. K., Shanna, G. L. & Ali,, M. (1987). Plant based anriamoebic drugs. Parr I. Anuamoebic activity of phenanrhroindolizine alkaloids; common structural de-
524
terminants of activity with emetine. Planta Medica, 53, 532-536. Blanco, A., Aoki, A., Montamat, E. E. & Rovai, L. E. (1983). Effect of gossypol upon motility and ultrastructure of Typanosoma cruzi 3ournal of Protomology, 30, 648-651. Campbell., W. C. (1986). The chemotherapy of parasitic infections. Journal of Parasiwlogy, 72, 45-61. Chang, M. C., Zhiping, Gu. & Saksena, S. K. (1980). Effects of gossypol on male rats, hamsters and rabbits. Contraception, 21, 461-469. Chatterjee, D. K., Iyer, N. & Ganguli, B. N. (1987). Antiamoebic activity of chonemorphine, a steroidal alkaloid, in experimental models. Parasiwlogy Research, 74. 30-33. Chintana, T., Sucharit, P., Mabakittikun, V., Siripanth, C. & Suphadtanaphongs,W. (1986). In vitro studies on the sensitivitv of local Entamoeba hiswlvtia to anti-amoebic drugs. S&&east Asian 3oumal of &pica1 Medicine and Public Health, 17, 591-594. Dorsett, P. H. & Kerstine, E. (1975). Antiviral activity of gossypol and apogossypol. 3oumal of Pharmaceutical Sciences, 64, 1073-1075. Finney, D. L. (1977). Probit Analysis. New York: Cambridge University Press. Gonzalez-Garza, M. T. & Said-FemBndez, S. (1988). Entamoeba hisw&ica: potent in vitro antiamoebic effect of gossypol. Experimental Parasiwlogy, 66, 253-255. Jensen, D. R., Sorensen, J. M. & Bosek, S. A. (1982). Deposition pattern of antifertility agent, gossypol, in selected organs of male rats. Toxicology, 24, 65-72. Keene, A. T., Phillipson, J. D., Warhurst, D. C., Koch, M. & Seguin, E. (1987). In tifro amoebicidal testing of natural products. Part 2. Alkaloids related to emetine. Planta Medica, 4, 201-206. Liu, G.-z., Ch’iu-Hinton, K., Cao, J., Zhu, C. & Li, B. (1988). Effects of K salts or potassium blocker on gossypol-related hypokalemia. Cuntracepfiun, 37, 11l_.III. Montamat, E. E., Burgos, C., Gerez de Burgos, N. M., Rovai. L. E. & Blanco. A. (1982). Inhibitorv action of gossy& on enzymes &d grbwth’of Trypanokma cnui. Science, 218, 288-289. Neal? R. A. (1978). Antiamoebic activity of drugs given smgly and in combination against axenically grown Entmweba hiswlvtica. Archives de Znvestipaci& MLdica (Mexico), 9, (subplement), 387-392. -
Neal, R. A. (1983). Experimental amoebiasis 9d developTp;:9;f anti-amoebic compounds. Paraslwlogy, 86, Radloff, R. ‘J., Deck, L M., Royer, R. E. & Vander Jagt, D. L. (1986). Anti&al activities of gossypol and its derivates against herpes simplex virus type II. Phannacological Research Communications,18, 1063-1073. Rikihisa, Y. & Lin, Y. C. (1986). Taenia taeniaefmis: inhibition of metacestode development in the rat by gossypol. Experimental Parasiwloa, 61, 127-133. Royer, R. E., Deck, L. M., Campos, N. M., Hunsaker, L. A. & Vander Jagt, D. L. (1986). Biologically active derivates of gossypol: synthesisand antimalarial activities of peri-acylated gossylic nitriles. 3oumal of Medicinal C&ma&y, 29, 1799-1801. Said-Femhdez, S., Vargas-Villareal, J., Castro-Garza, J., Mata-Cardenas, B. D., Navarro-Marmolejo, L., LozanoGarza, G. & Martinez-Rodriguez, H. (1988). PEHPS medium: an alternative for axenic cultivation of EnTransact@ of the tamoeba hiswlytica and ,E. invad?. f4~jLociery
of Troprcal Medmne and Hygaene, 82,
Van der Jab, D. L., Heidrich, J. E., Royer, R. E. & Hunsaker, L. (1982). Antimalarial activity of gossypol and methylglyoxal-bis-guanylhydrazone. Federation Proceedings, 41, 1428. Wang, M. Q., Wu, E. F. & Yu, Y. W. (1985). Highperformance liquid chromatography with electrochemical detection of gossypol in human plasma. 3o~nal of Chromarography and Biochemical Applications, 343, 387-
3%. Wickmann, K., Vaheri, A. & Luukkainen, T. (1982). Inhibition of herpes simplex virus type 2 infection in human epithelial cells by gossypol, a potent spermicidal and contraceptive agent. American Journal of Obstetrics and Gynecology, 142, 593-594. Xu, D., Cai, W.-j., Zhu,B.-j., Dong, C.-j., Zheng, Z.-c. 81 Gao?Z.-q. (1988). Clinical safety of long-term administranon of gossypol in 32 cases. Contraception, 37, 129-135. Zar, J. H. (1974). Btistatistical Analjrsis. Englewood Cliffs, New Jersey: Prentice-Hall, pp. 228-235.
Received 24 October 1988; accepted Januuy 1989
for publication
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