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.The collection ofplants relied heavily on taxonomy, in both the selection of plants for collection,screening and identification of active compounds in the crude extract.Anexample of the advantages of the use of toxonomy in identifying knownactive compounds are as follows: a collection of Merrilliodendron megacarpum(Icacinaceae) showed high activity against the P 388 leukemia, and a literaturesurvey revealed that the plant has not been chemically investigated.The only 92 Bioactive Marine Natural Productshighly active compound previously isolated from Icacinaceae was camptothecinwhich was isolated from Nothapodytes foetida (syn.Mappia foetida).Comparison of the crude extract of M.megacarpum with a sample ofcomptothecin led to tentative identification of camptothecin in the crudeextract of the plant.The identity of the compound was finally confirmed byisolation, thereby saving considerable time and expense over bioassay directedisolation.The screening programme of the CCNSC since its inception was based onrandom screening of plants, that is, plants that have no reason for beingscreened other than novelty.There was fairly extensive literature on folkloreand traditional medicine in the treatment of cancer.43 Spjut et al44 did aretrospective correlation of several groups of medicinal and poisonous plantswith the NCI screening data and found substantially higher activity for plantsused as anthelmintics, arrow poisons and fish poisons when compared withplants selected randomly.Further examination of the data indicated that thehighest correlation was between poisonous or toxic plants and in vitro activity(cell toxicity), and that the correlation between these plants and activity invivo was not high.It was not surprising since poisonous plants are toxic tocells in culture.4.2 In vitro and in vivo ActivityThere is a great deal of loose usage of terminology with reference to in vitroand in vivo anticancer activity as pointed out by Suffness et al.42 Manycompounds are cited as anticancer or antitumour agents, which are, in fact,only cytotoxic to tumour cells in vitro.This crops of compounds contains awide variety of toxic substances which display no particular selectivity towardstumor cells as opposed to normal cells and have no hope of being usefulanticancer agents.Suffness et al42 has suggested the following terminologyto eliminate the confusion.The term cytotoxicity means toxicity to tumourcell in culture.The term anticancer, antitumour or antineoplastic, therefore,should not be used to express in vitro testing results.For in vivo activity inexperimental system, it is suggested to use the term antitumour orantineoplastic in reporting results.42 Besides, the term anticancer should bereserved for reporting clinical trials data in man.4.3 Screening MethodsThe quality of the screening methodology employed is the most importantfactor in drug discovery for any type of biological activity.The predictabilityof the screens for clinical activity is absolutely critical, since if the screensidentify compounds which are clinically inactive, all the efforts which helpthe development of such compounds are wasted.The correlation of screening actives with clinical activity is an extremely difficult process since it takesa number of years from when active compounds are tested until results ofclinical trials are available, and the data base is very small since few compoundsultimately reach clinical trials. Biological, Toxicological and Clinical Evaluation 93New screens are usually validated by testing a wide variety of agentswhich have been under clinical trials and comparing the detection of knownclinically active compounds (true positives) with the detection of compoundswhich show activity in experimental screens, but not in man (false positives)and compounds which are clinically active but undetected in experimentalscreens (false negatives).The data thus, obtained can be used to judge thequality of the new screen.4.4 Screening ProblemsThere are special problems encountered in screening extracts of plants andanimals.Generally, the active principle present in the crude extracts arepresent in very low concentration, therefore, the screen for their detectionshould be very sensitive, in general, in vitro test methods are more sensitivethan in vivo method.Besides, screen should be selective, and specific.Themethodology must be adaptable to materials which are highly coloured,tarry, poorly soluble in water and chemically complex.The second problemin screening crude extracts is that the pre-screen, screen or bioassay usedmust be insensitive to the inactive compounds which are potential interferingsubstances.The assay must also be insensitive to ubiquitous compoundswhich will give false actives.The NCI dropped the Walker 256 model whilescreening extracts of plants since it was highly sensitive to tannins.Theassay also must be highly selective.The assay chosen must still meet theother requirements of any good assay, including validity, predictability,correlation, reproducibility, and the cost should be reasonable.4.5 Current Approach and StatusThe current approach at the NCI for screening crude extracts is to use aprimary in vivo assay, the P 388 leukemia, and to use in vitro systems asbioassay to guide isolation of in vivo active compounds.The advantages ofa battery of pre-screens are that (i) there is a greater chance to detect novelcompounds; (ii) compounds with differing mechanisms of action can beselected and (iii) identification of known compounds is simplified since onlycertain classes of compounds will show activity in particular pre-screens.The biologically active compounds are generally present in low concentrations(1 to 100 mg per kg of dried plant), and the chances of finding these compoundsby the standard phytochemical method of purifying and characterising themost abundant compounds present in an active extract are quite rare.Thebioassay guided isolation method is essential in these cases.The major bioassayscurrently in use in the NCI programme are the KB and P 388 cell lines.While these in vitro systems are excellent bioassays, they are poor screensbecause of their sensitivity to cytotoxic substances which are devoid of invivo activity.All novel compounds which are reproducibly active in the P388 leukemia pre-screen then put to tumour panel testing [ Pobierz całość w formacie PDF ]
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.The collection ofplants relied heavily on taxonomy, in both the selection of plants for collection,screening and identification of active compounds in the crude extract.Anexample of the advantages of the use of toxonomy in identifying knownactive compounds are as follows: a collection of Merrilliodendron megacarpum(Icacinaceae) showed high activity against the P 388 leukemia, and a literaturesurvey revealed that the plant has not been chemically investigated.The only 92 Bioactive Marine Natural Productshighly active compound previously isolated from Icacinaceae was camptothecinwhich was isolated from Nothapodytes foetida (syn.Mappia foetida).Comparison of the crude extract of M.megacarpum with a sample ofcomptothecin led to tentative identification of camptothecin in the crudeextract of the plant.The identity of the compound was finally confirmed byisolation, thereby saving considerable time and expense over bioassay directedisolation.The screening programme of the CCNSC since its inception was based onrandom screening of plants, that is, plants that have no reason for beingscreened other than novelty.There was fairly extensive literature on folkloreand traditional medicine in the treatment of cancer.43 Spjut et al44 did aretrospective correlation of several groups of medicinal and poisonous plantswith the NCI screening data and found substantially higher activity for plantsused as anthelmintics, arrow poisons and fish poisons when compared withplants selected randomly.Further examination of the data indicated that thehighest correlation was between poisonous or toxic plants and in vitro activity(cell toxicity), and that the correlation between these plants and activity invivo was not high.It was not surprising since poisonous plants are toxic tocells in culture.4.2 In vitro and in vivo ActivityThere is a great deal of loose usage of terminology with reference to in vitroand in vivo anticancer activity as pointed out by Suffness et al.42 Manycompounds are cited as anticancer or antitumour agents, which are, in fact,only cytotoxic to tumour cells in vitro.This crops of compounds contains awide variety of toxic substances which display no particular selectivity towardstumor cells as opposed to normal cells and have no hope of being usefulanticancer agents.Suffness et al42 has suggested the following terminologyto eliminate the confusion.The term cytotoxicity means toxicity to tumourcell in culture.The term anticancer, antitumour or antineoplastic, therefore,should not be used to express in vitro testing results.For in vivo activity inexperimental system, it is suggested to use the term antitumour orantineoplastic in reporting results.42 Besides, the term anticancer should bereserved for reporting clinical trials data in man.4.3 Screening MethodsThe quality of the screening methodology employed is the most importantfactor in drug discovery for any type of biological activity.The predictabilityof the screens for clinical activity is absolutely critical, since if the screensidentify compounds which are clinically inactive, all the efforts which helpthe development of such compounds are wasted.The correlation of screening actives with clinical activity is an extremely difficult process since it takesa number of years from when active compounds are tested until results ofclinical trials are available, and the data base is very small since few compoundsultimately reach clinical trials. Biological, Toxicological and Clinical Evaluation 93New screens are usually validated by testing a wide variety of agentswhich have been under clinical trials and comparing the detection of knownclinically active compounds (true positives) with the detection of compoundswhich show activity in experimental screens, but not in man (false positives)and compounds which are clinically active but undetected in experimentalscreens (false negatives).The data thus, obtained can be used to judge thequality of the new screen.4.4 Screening ProblemsThere are special problems encountered in screening extracts of plants andanimals.Generally, the active principle present in the crude extracts arepresent in very low concentration, therefore, the screen for their detectionshould be very sensitive, in general, in vitro test methods are more sensitivethan in vivo method.Besides, screen should be selective, and specific.Themethodology must be adaptable to materials which are highly coloured,tarry, poorly soluble in water and chemically complex.The second problemin screening crude extracts is that the pre-screen, screen or bioassay usedmust be insensitive to the inactive compounds which are potential interferingsubstances.The assay must also be insensitive to ubiquitous compoundswhich will give false actives.The NCI dropped the Walker 256 model whilescreening extracts of plants since it was highly sensitive to tannins.Theassay also must be highly selective.The assay chosen must still meet theother requirements of any good assay, including validity, predictability,correlation, reproducibility, and the cost should be reasonable.4.5 Current Approach and StatusThe current approach at the NCI for screening crude extracts is to use aprimary in vivo assay, the P 388 leukemia, and to use in vitro systems asbioassay to guide isolation of in vivo active compounds.The advantages ofa battery of pre-screens are that (i) there is a greater chance to detect novelcompounds; (ii) compounds with differing mechanisms of action can beselected and (iii) identification of known compounds is simplified since onlycertain classes of compounds will show activity in particular pre-screens.The biologically active compounds are generally present in low concentrations(1 to 100 mg per kg of dried plant), and the chances of finding these compoundsby the standard phytochemical method of purifying and characterising themost abundant compounds present in an active extract are quite rare.Thebioassay guided isolation method is essential in these cases.The major bioassayscurrently in use in the NCI programme are the KB and P 388 cell lines.While these in vitro systems are excellent bioassays, they are poor screensbecause of their sensitivity to cytotoxic substances which are devoid of invivo activity.All novel compounds which are reproducibly active in the P388 leukemia pre-screen then put to tumour panel testing [ Pobierz całość w formacie PDF ]