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Preclinical Screening and Evaluation of Agents for the Chemotherapy of Cancer- A Review* ABRAHAM GoH)m, JOHN M. VENDITTI, AND NATHAN MANTEL (Laboratory of Chemical Pharmacology, and Biometry Branch, National Cancer Institute, Bethesda, Md.) The current extensive programs on antitumor chemotherapeutic screening and drug evaluation are conducted mainly in rodents with transplantable tumors (7, l l , 13, 14, 19, ~4, ~6, 43, 45, 46, 48, 56, 57, 60-65, 67, 7~, 75, 78, 84-91, 94, 99). Additional programs are being conducted with spontaneous and carcinogen-induced tumors (1~, 48, 69, 79, 80, 96) and with tissue culture (8, 17, 18, ~1, ~ , 47, 59, 88, 95), microbiological (1, 5, 6, 16, 20, el, e3, 50-53, 55, 77, 8~, 88, 9~), and biochemical (9, 15, 49, 66, 71, 8~) systems, and systems involving development and differentiation (3, 58, 81), etc. Numbers of compounds are passing the screening and evaluation tests and are being introduced into preclinical and clinical pharmacology and into clinical trial. There is considerable interest in the question of how well the screening and evaluation tests may predict clinical usefulness (~tS, 73, 75, 83). Will activity in specific animal tumor systems correlate well with activity in specific types of human tumor systems? Can the animal systems not only detect antitumor activity, but also rate drugs with respect to such activity ? Will the rating of the drugs in the animal systems correlate with the comparative therapeutic evaluation of antitumor effectiveness in man (10~)? Basic to any attempt to determine correlation of animal testing and chemotherapeutic effectiveness in humans is the validity of the animal screen or evaluation test itself. What information does the animal screen or test provide? Is it actually evaluating activity in the animal system? Is it appropriately ordering the compounds in terms of relative effectiveness? Are the criteria of measurement of therapeutic effect appropriate? ks the assay system paying sufficient attention to the hosttumor-drug relationship? This basic question is * The authors are indebted to Dr. E. K. Marshall, Jr., for his stimulation, interest, and many helpful suggestions in the undertaking of this review. Received for publication March ~0, 1961. prompted by the work of E. K. Marshall (68), who pointed out that any evaluation of agents in infection chemotherapy should take into account the triad of parasite, host, and drug. The current review was undertaken to determine what information the common animal screening and evaluation procedures may provide. The parameters of drug action examined are those employed in common practice. In the analysis no attempt is made to be exhaustive, but rather to review the types of coinman screening and evaluation procedures and their meaning. I t is hoped that this may contribute to the re-examination of ways and means of improving animal and related screens, so t h a t any attempt at correlation with results in humans may be more meaningful. T U M O R I N H I B I T I O N ASSAYS In the study of the antitumor activity of drugs, one may determine the effect of the drug on the tumor without any regard to its effect on the host. This procedure conceivably could be useful, if the antitumor agents were nontoxic for the host. In such case, the evaluation of drug effectiveness could be limited to the determination of the amount of drug required to produce a desired degree of inhibition or cure of the tumor. Or the evaluation could be based on the time or number of injections required to produce a specified antitmnor effect, or the rapidity of occurrence of a refractory state, or the degree of activity for a spectrum of tumors, etc. Assay systems could, in the above cases, be limited primarily to simple dose-response studies with respect to the tumor, in the main ignoring effects of the drugs on the host. Although the drugs might differ in physiological disposition, including transport, excretion, or metabolic alteration, etc., host toxicity would be removed as a factor in the assay. The methodology would be similar to t h a t employed in the in vitro tissue culture and bacterial systems, and the extent of correlation of results with these systems could be determined. 1334 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. GOLDIN el al.~Preclinical Screening and Evaluation From the point of view of therapeutic applicability, assay systems of drug effectiveness based on antitumor effect alone pertain to too simple a situation, since they presuppose that antitumor agents are nontoxic for the host. All of the known antitumor agents are toxic for the host as well as for the tumor, and the toxicity for the host severely limits their usefulness. If the tumor system employed for assay is extremely sensitive to drug treatment this could, in effect, remove host-toxicity as a factor. In such ease, since relatively nontoxic doses would exert antitumor effect, it might still be possible to limit the assay to evaluation of antitumor action of the drug. Such a system may be provided by using a tumor system empirically found to be sensitive, or selection could be practiced for sensitivity. A tumor system could also be made more sensitive by diminishing the size of the inoeulum (41, 44) or by initiating treatment at an early time following tumor implantation (~7, 135, 40, 78, 98). Such a system could then be employed to compare the relative effectiveness of drugs in inhibiting tumor growth (57, 78, 98). In some instances, it may be desirable to employ a relatively sensitive tumor system for determining marginal antitumor effects of drugs. This may have a practical value-for example, in screening for drugs to be employed as surgieat adjuvants. IIowever, care must be exercised in the utilization of such assay systems. If two drugs completely inhibit early tumor growth at nontoxic doses, which is the more effective drug? I t could, presumably, be the one, for example, which accomplishes complete inhibition at a lower dose. However, this might indeed be misleading if neither drug were capable of inhibiting tumor growth in mice with more advanced tumor. Since the known antitumor agents are limited in their usefulness because of their toxicity for the host, an assay system in which drug toxicity for the host is limiting may more closely reflect the clinical situation. A standard procedure employed in screening programs has been to measure the extent of inhibition of growth of the local transplanted tumor after a specified number of treatments. Usually the animal is sacrificed and the tumor is enueleated and weighed. Alternatively, the local tumor may be measured in one or more dimensions. Comparison is then made with the size or weight of the tumor for untreated control mice. The per cent inhibition of tumor growth is thus determined and must usually exceed a specified figure in order for a drug to pass a primary screen. The ratio t/c (fractional growth of treated tumor as compared with control) is commonly 1335 employed, where t is the average weight or size of treated tumors and c is the control average. In order to take into account nonspecific toxicity for the host it is usually required that t/c must not only be less than a specified fraction, but that this must be achieved with a limited animal weight loss and/or a limited host mortality response. The above type of procedure has been employed in primary screening programs to determine whether drugs are of sufficient interest for further study (7, 12, 19, ~4, ~6, 61-65, 7~, 84, 85, 87, 89, 90, 94). By such a test system, the compounds may be ordered with respect to the relative degree of effectiveness; i.e., comparisons may be made of t/c at a maximum tolerated dose. Where both host effect and tumor effect are to be taken into consideration, the question arises as to the choice of parameters for each. Toxicity for the host is usually measured in terms of drug lethality, or as body weight loss. The measure of antitumor effect may be based on tmnor size or weight, or on some other characteristic of the tumor, such as protein content, etc. Cytotoxie effects or biochemical effects may be measured. The growth curve of the tumor may provide a useful measure of antitumor effect. Survival time of all mice or, alternatively, only of mice not succumbing to drug toxicity, have been used as measures of drug effectiveness. Where drug effects are extensive, or the tmnor challenge weak, the percentage "cures" elicited may provide an index of drug effect. SURVIVAL T I M E ASSAYS Whereas routine screening procedures most commonly employ tumor size or tumor weight as the basis for evaluating antitumor effect, there are some notable exceptions. For example, in chemotherapeutic screening with the LI~10 tumor, the Cancer Chemotherapy National Service Center employs an index of effect based on the prolongation in survival time of tumor-bearing mice (6165). Survival time as a measure of chemotherapeutic effect has been used by various investigators in laboratory research with the tumorous host (11, 13, 14, 24, 48, 56, 57, 67, 69, 78, 80, 86, 96). Since increasing survival time, rather than reducing tumor size, more properly reflects the purpose of treatment, such extension would, in many circumstances, more appropriately describe the therapeutic efficacy of treatment. Accordingly, in this laboratory, assay systems have been developed in which alternative ehemotherapics are compared on the basis of the survival time of the tumorous host (~27, 81, 40, 43-46, 99). Since, for each drug on a specified treatment schedule, survival time varies with dose level, an appropriate measure for Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. Cancer Research 1836 T U M O R SIZE VS. S U R V I V A L T I M E If extension in the survival time of tumor-bearing animals is the motivation of experimental chemotherapy, justification for using inhibition in tumor size as a criterion, aside from perhaps its simpler ascertainment, would be dependent upon the existence of an underlying correlation between the two measures of effectiveness. The question, then, is whether or how tumor inhibition can be used to select agents capable of achieving important increases in survival time. a drug on a schedule is the maximum survival time it elicits. At dosages beyond some optimal level, toxic effects for the host outweigh antitumor effects so t h a t further increases in dosage lead to reductions rather than extensions in survival time. Use of the maximum survival time elicited with a drug on a schedule as the measure of therapeutic effect leads to certain modifications in laboratory procedure. To ensure bracketing the optimal dose level, a relatively wide dosage range should be employed. Also, the interval between successive <~ OC MT___X N T 9 _~ 3 0 0 > L " : 9 Z ! T' -- 0 [ 6-MP_ __6-THIOOUANINE AZASERINE " <_2o '~ Vol. ~1, N o v e m b e r 1961 9 | : , : , - - : . . . . . t 9 " : /. J I l I I 0.4 0.9 2_.I I ] I I 9 - : 1210 ~ / = 9 [,/: I LEUKEMIA , . :/ I I I I I I . . I i . I . i . ! 1.4 3.1 7.0 16 35 60 160 DAILY DOSE, M S / K S . 1 . l ] 24 I I I I 53 120 CttART 1.--Illustration of use of survival time assay employing advanced leukemia LI~10 in mice (see reference 43). In this experiment treatment was initiated 9 days following tumor inoculation, this being only 3 days before the median day of death (day 1~) of untreated controls. The optimal dose of methotrexate (MTX), 0.9 mg/kg, yielded a median survival time of ~6 days, 17 days beyond the initiation of treatment. With 6-thioguanine, the optimal dose was 7.0 mg/kg, the median survival time being 19.5 days. 6-Mercaptopurine (6-MP) at a dose level of 180 m g / k g yielded a median survival time of ~0 days, whereas 53 m g / k g daily of azaserine yielded a median survival time of 17 days. The respective maximum increases in doses must be small enough so t h a t the observed optimal dose and maximal survival time may be taken as near approximations to their unknown true values. In employing this methodology, deseribed in detail in other publications (40, 43-46, 99), it has been the praetiee of the authors to use the median survival time of a group of mice as the summary measure of survival time. Individual survival times may, nevertheless, be reported (45, 46, 99), protection thus being afforded against instances where the median survival time may be misleading. Chart 1 provides an illustration of the use of this procedure for determining the relative effectiveness of several antileukemie agents. There are two aspects to this question. First, where an agent can, in fact, elicit important increases in survival time, or possibly even cures, how likely is this agent to be detected in a tumorinhibition assay? Second, to what extent m a y agents, capable of eliciting only limited, nonspecific increases in survival time, give rise to reductions in tumor size? These aspects will be discussed below. Where the use of tumor inhibition is premised on its being an easily ascertainable basis for selecting effective agents, the question of how much more efficient tumor-inhibiton assays m a y be also arises. Goldin et al. (44) have suggested that, median survival time over untreated controls are: methotrexate, 14 days; 6-thioguanine, 7.5 days; 6-MP, 8 days; azaserine, 5 days. Note that, although some control mice died within a day or two after the scheduled time of initiation of treatment, such early deaths did not occur among mice receiving effective levels of methotrexate. In this experiment, treatment was discontinued for the last two survivors in each group, to provide the possibility for identifying cures resulting from previous treatmerit. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. Got, DISC et al.~Preclinical Screening and Evaluation under certain circumstances, survival time assays can be performed with efficient use of time and animals. In such case, tumor-inhibition assays may be unnecessary. Although the emphasis in survival time assays is on the longevity of treated animals, it is not intended that in such assays toxic manifestations should be overlooked. One would obviously be dubious about accepting as an effective agent one which, while increasing survival time, resulted in permanent injury to a vital organ. Assays to determine effectiveness of agents.--It is perhaps naive to expect too high a correlation between the tumor-inhibitory effects of agents and their potential for extending survival time. Measurement of inhibition of tumor growth does not permit accurate extrapolation of survival time. Factors which may influence the relationship of inhibition of tumor growth to survival time include the drug dosage, schedule of therapy, and the nutritional status of the host. The relationship may vary in accordance with the time at which the observations are made. Agents effective in increasing survival time nmy achieve such prolongation in a number of ways. For an effective agent with a true tumorieidal effect a correlation might be readily obtained between tumor inhibition and survival time, provided the chemical agent is relatively nontoxic for the host. The correlation would be less readily demonstrable if the agent is toxic for the host. Alternatively, an agent may behave as a eareinostat, perhaps even with stasis not being evident until after the time selected for sacrifice of animals in a tumor-inhibition assay. While eareinostasis might lead to increased longevity, it would not necessarily produce effects detectable in a tumor-inhibition assay. There may be agents capable only of inhibiting invasion and metastasis of the neoplastic process without affecting the primary tumor; here the infltrating tumor would appear to be sensitive and the local tumor resistant. In such instance, an agent could be overlooked readily in a tumor-inhibition assay. Conversely, there may be agents which inhibit the primary tumor but fail to retard metastatic infiltration. Increase in survival time of mice with leukemia L1210 has been observed to occur with either progressive growth of local tumor at the subcutaneous site of inoculation of leukemic cells or a decrease in size or actual disappearance of the local tumor (131). If agents producing the kinds of effects described above were encountered, restriction of measurement to observation of local tumor size should be particularly hazardous as a measure of therapeutic effectiveness. 1337 When one considers results obtained with a range of dose levels, some difficulties can be visualized. For an effective drug, as the dose is increased, the rate of tumor growth may be diminished and the survival time increased. It is clear, however, that, owing to toxicity for the host, the very highest dose levels may give the highest degree of tumor inhibition, yet show the shortest survival times. This difficulty can be met in part by requiring that the dosage level employed be one that yields little lethality (or toxicity), at least as of the time of sacrifice. This may only yield fresh complications, due to differences among agents in the time at which they cause death or other toxic manifestations. With some agents, animals which have received lethal treatment may still be alive and in apparently good condition as of the scheduled date of sacrifice. Tumor inhibition would here be ascertained at what is, in fact, a nontolerated dose. Further difficulties arise when one considers the possibility of cumulative toxic effects for the host. Where cumulative effects are minimal, one may continue to treat successfully animals which have not yet shown important tumor inhibition. Conversely, the greater inhibitory effect for another agent may be accompanied by such cumulative toxic effects for the host as to make impossible further successful treatment. It is important to emphasize that, in the employment of survival time assay systems, one is guarded against the above types of situations. A variety of relationships encountered between tumor inhibition results and survival time extension is illustrated in Charts ~a, b, 3a, b. Nonspecific effects of treatment.--Nonspecific drug toxicity for the host, including reduced caloric intake and body weight loss, may alter tumor growth and survival time. There need be no precise correlation between the effect of nonspecific toxicity on tumor size and survival time. Inhibition of tumor growth by caloric restriction may or may not result in an increase in survival time, or it may, if the restriction is too extensive, actually result in a decrease in survival time. With increasing weight loss there may be progressive inhibition of tumor growth, but survival time may increase with moderate weight loss and decrease with excessive weight loss. Various investigators (r 4, ~8, 54, 76, 80, 93,101) have produced tumor inhibition by caloric restriction. In one series of experiments it was shown that the degree of tumor inhibition of Sarcoma 180 elicited with a variety of agents corresponded to that obtained with levels of caloric restriction giving the same weight loss (~8). This Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. I I I I I 1 I I I I (~ARONOM^ 755 - CHEMOTHERAPY,DAYS 4 THRU8 (}-DAILY TREATMENTLEVEL IN MG/KG. 3 0 - I-MEDIAN SURVIVALTIME 25 35 30 ~E "~ 2 5 uZ CHEMOTHERAPY4--- CARCINOMA 755 _i _] ~: I - MEDIAN SUR-~-i (c) -~ ~. I 0 6 - MP ,~/,.--I (c} -I (~] (sg) 20 - - N 20 // tD _z15 _> . 4 >_ C:YTO ~:t0 c~ "o 5 / ,,,0 , , , , , , , ' ' CYTOXAN _J ~30 I (leO) NO TUMORS DEFINITE O :~ 2 5 - ,,, 2 0 -t ,,,15 J, I0 I (50) 0' 0 I I I0 I I I 20 5 l 30 i l 40 i I .J 50 DAYS POST TUMOR INOCULATION O0 DEFINITE TUMORS ~ (8.3) uP TO DAYlfl ~ t I0 ', 20 i i 30 i i i 40 I J 50 DAYS POST TUMOR INOCULATION C~ART 2a C}~A~m 2b ~2a AND ~b.--Examples of experimental antitumor activity employing Carcinoma 755. These charts permit following the progressive antitumor effects of treatment and show also its therapeutic efficacy as evidenced by a prolongation in survival time. Each curve shows tile average tumor diameter, as measured by palpation, of a group of treated mice on specific days following tumor implantation. The curves are discontinued on the day of median survival of the group. For the experiment represented by Chart 2a, daily treatment with graded dosages of compound was initiated 4 days following tumor inoculation and was continued until death of the animals. It can be seen that both 6-mercaptopurine (6MP) and Cytoxan were effective at the optimal dosages of 8.1 and 6.~ m g / k g daily, respectively, in increasing the median survival from ~4 days for untreated controls (C) to 50 days. With Cytoxan this increase in survival time was accompanied by only a moderate tumor-inhibitory effect, contrasting with the marked inhibition due to 6-MP. Itad tumor inhibition at some early sacrifice date been the only criterion for evaluating an agent, standards by which 6-MP would have been accepted could well have led to the rejection of Cytoxan, an equally effective drug by survival time standards. It m a y be noted, especially for the Cytoxan data, that doses beyond the optimal, while showing increased tumor inhibition, lead to decreased survival times. For the experiment represented by Chart el), daily drug treatment was initiated on day 4 and continued for only 5 days. With this curtailed treatment the optimal daily dosage was greatly increased, to 65 m g / k g for 6-MP and to 108 m g / k g for Cytoxan. Again it is evident that the efficacy of 6-MP (greater this time than that of Cytoxan) is accompanied by marked tumor-inhibitory effects, these being so great that tumors are not evident for an extended period following the discontinuance of treatment. Overdosage with 6-MP to a level of 180 m g / k g daily resulted in a decrease in survival time. ~,5-Bis(1-aziridinyl)-g,6-bis(~2-methoxyethoxy)-p-beI~zoquinone (A-159) which elicited clear tumor inhibition at a daily dosage of 5 m g / k g did not produce any important extension in survival time. In other experiments with Carcinoma 755, not shown here, treatment with 6-MP or with Cytoxan was not initiated until as late as day 13, at which time mice already had clear palpab]e tumors. Drug treatment at this time, which was effective in extending survival time, caused the tumors to regress, though ultimately there was regrowth. CIIART$ Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. GOLDIN et al.--Preclinical Screening and Evaluation is not to say that the antitumor effect of the agents was necessarily mediated through a food restriction mechanism. However, it does suggest that the effects observed were nonspecific, reflecting only damage occurring to the host. Toxic effects for the host are generally evident from the over-all weight loss which follows. Where the nonspecific effect on a tumor is due to a toxic effect not reflected in over-all host weight loss, the effect may mistakenly be taken as real. Alternatively, real antitumor effccts which happen to be accompanied by weight loss can be mistaken as nonspecific. When caloric restriction is cmployed as t h e n o n s p e c i f i c a g e n t , t u m o r - i n h i b i t o r y effects exI I I i I I I I 1339 tensive enough to meet most screening requirements can be achieved. However, these effects will, in general, be accompanied by substantial weight loss. The relationship of tumor inhibition and survival time of the animals m a y be dependent upon t h e e x t e n t of u n d e r f e e d i n g . T h e r e l a t i o n s h i p m a y change with the time at which the measurements of t u m o r size are t a k e n . T h e s c h e d u l e of u n d e r feeding, including the time at which underfeeding is t e r m i n a t e d , m a y also i n f l u e n c e t h e r e l a t i o n s h i p , etc. A l t e r a t i o n of d i e t m a y also a l t e r t u m o r g r o w t h s u r v i v a l t i m e r e l a t i o n s h i p s , etc. W i t h f e e d i n g of 2~ ] I -- [ T~- ! - ~ - - ; j J (o.2~) '(c) NITROGEN ( f o . : , (o.~o) MUSTARD. ) 9---~(z.o) o s- N 0 z I I - SARCOMA 37 CHEMOTHERAPYFROM DAY3( ) - DALLY TREATMENT LEVEL IN MG/KG. I ,,' MEDIAN SURVIVAL TIME ] I (9.g) m 20- { I t .,~r~ I0 -- SARCOMA 37 CHEMOT~ ~3 THRU 7~ ()=DAILY TREATMENT LEVEL i IN MG/KG I - MEDIAN SURVIVAL TIME {/// r | 5 z_ I oc HYDROCORTISONE (c) L :~:~ 0 6-THIOGUANINE 2 t I I (r o I5_ ,.,. ! 7 --I (5 o) ta..i J ~0 < e-~ 5 o :~ 0 ~ ~, I I I I I I I (39l I i t (c) k- e--- 5 t-- ( 4 - APP 9 ,., 20 ,,, 2 0 < -4 (c) ~9 // t~ ~ I0 "~ 10 --I (333) 5 N-METHYL FORMAMIDE 0 4 I ! ' ' I ', ! ! 8 12 16 20 24 28 32 36 DAYS POST TUMOR INOCULATION CHART - 0( 40 33. Caxars 3a AND 3b.--Examples of experimental antitumor activity employing Sarcoma 37. For explanation of how results are shown in these charts see legend for Charts 2a and ~b, first paragraph. For the experiment represented by Chart 3a, daily treatment with graded dosages of compound were initiated 3 days following tumor inoculation and were continued until death of the animals. No increase in survival time was elicited with either nitrogen mustard or hydrocortisonc. Ovcrdosages with these compounds which reduced survival time did not generally produce tumor-inhibitory effects. At an optmml daily dose of 1~20mg/kg, N-methyl formamide yielded a median survival time of ~5 days, compared with 17.5 days for untreated controls (C). This was not accompanied by important tumor inhibition. The overdose of 333 mg/kg daily yielded much +(3 9) 5t o 4 : ! I ! ~ 1 I 8 12 16 20 24 28 32 36 DAYS POST TUMOR INOCULATION C~AR'r 40 3b more extensive tumor inhibition, but survival time was reduced. For the experiment summarized in Chart 3b, daily treatment with 8-azaguanine, 6-thioguanine, or 4-aminopyrazolo(3,4-d)pyrimidine (4-APP) was initiated on day 3 and continued for only 5 days. None of the three compounds was effective in increasing survival time, and only one, 6-thioguanine, gave evidence of tumor inhibition. For the other two compounds, antitumor effects were not clearly evident even at overdosages. In Charts ~a, ~b, 3a, and 3b, a variety of relationships between drug effects may be seen. Drug efficacy in extending survival time may be accompanied by a greater or less degree of tumor inhibition. Drug toxicity for the host may or may not be accompanied by tumor-inhibitory effects. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. Cancer Research 1340 Vol. ~1, N o v e m b e r 1961 desiccated tumor, the Walker tumor continued to grow, and survival time was increased (70). The age and weight of the mice may also alter tumor growth-survival time relationships (30, 36). In general, where survival time is the end-point employed, the problem of nonspecific effects is likely to be less critical. Work in this laboratory has shown that, while caloric restriction may produce important reductions in tumor size, the increases in survival time to which it gives rise may tend to be less dramatic (54). In view of the drastic effects that diminished food intake may have on the growth of transplantable tumors, and the possibility of underfeeding actually increasing survival time, the importance of characterizing experimental tumor systems with respect to the influence of underfeeding cannot be overemphasized. If the experimental tumor system is sensitive to underfeeding, and/or weight loss, what would it behoove us to screen thousands of drugs, only to select as positive those drugs that depress appetite! Further testing would be required, in such cases, to determine to what extent eo~ diminished food intake could account for the ob.._1 0 served inhibition of tumor growth. Experiments rr" ISO-CALORIC involving paired feeding or isocaloric controls I-COMPOUND EXP. CONTROL Z would be required, and they are time-consuming. 0 9 2 (1.2 MG/KG) 0 (J It may be profitable in setting up chemothera9 2~ 2 (0.6 MG/KG) 1:3 peutic assay systems to establish regression lines 22 I-for the tumors employed, relating degree of under133 17 feeding to antitumor effect or degree of weight loss [] 9 .J 3 to antitumor effect. The antitumor effect could be 5 measured as tumor weight, survival time, etc. The s E I LI_ extent of correspondence of the drug-induced anti0 (1 I 15 tumor effect to the regression lines may then be ~-I00 qdetcrInincd. A close correspondence would be ins dicative of nonspecific antitumor action of the 80 j drug. rY The complication of nonspecific toxicity in o 60 2~ evaluation of tile antitumor effectiveness of a drag :D may frequently be overcome in large measure by ~- 40 appropriate adjustment of the assay procedure. If 7 it is found that the test system employed is too "k9' 20 REGRESSION sensitive to nonspeeifie calorie restriction or host Ct2 LINE toxicity, this may sometimes be remedied by inI I I I I 1 I a_ 0 creasing the inoculum level or withholding therapy +10 0 -I0 -20 -30 -40 until the tumor is more advanced. Thus, food rePERCENT CHANGE IN ANIMAL WEIGHT striction may inhibit local tumor growth and inCltART 4.--Illustration of a test for nonspecific inhibitory crease survival time of mice inoculated with leukeeffects of treatment with nitrogen mustards on Sarcoma 180. mia LI~10 when the restriction is initiated within TMs chart is taken from Goldin et al. (~28). The tumor-inhibiseveral days following leukemic inoculation. IIowtory effect for each treatment group (open tigure) and its isoever, when the disease is suftieiently advanced, caloric control (closed figure) is plotted against the percentage calorie restriction produces minimal inhibition of animal weight loss occasioned by treatment. The line shown on the chart is based on the average relationship observed to occur the local tumor growth and does not increase surbetween tumor weight and animal weight loss when such effects vival time (54). This obviates the need for further wcrc brought out in food restriction studies 028). In the prestesting of compounds (with isoealorie controls, ent chart it can be seen that tumor weights for drug-treated etc.) that are found active against advanced leumice are substantially similar to those for their isocaloric controls and that body weight loss for the host can account for kemia LI~10. I t removes the need to be concerned such tumor inhibition as has occurred. about any possible failure of correlation of effect Treatmeut was initiated one day after tumor inoculation of drng-imposed calorie restriction on tumor and continued daily for 5 days. The compounds and daily doses growth and survival time in attempts to interpret are coded as follows: specific drug effects. Charts 4 and 5 illustrate some (1.~ and 0.6 mg/kg) Mcthylbis (~-chloroethyl) amine of the points made in the foregoing, relative to the ~ N,N,N~,N'-tetrakis (~-chloro(4.4 Ing/kg) ethyl) cthylcncdiamine effects of food restriction. E:~ W i m i 17 8 5 1 15 Furfuryl-bis (/3-chloroethyl) amine Ethyl-bis (~-chlorocthyl) amine Isopropyl-bis (~-chloroethyl) amine Bis ($-chloroethyl) amine _O-Methoxyethyl-bis (fl-chloroethyl) amine ('2.4 m g/kg) (0.4 mg/kg) (0.S mg/kg) (~20.'2 mg/kg) (0.4 mg/kg) TIIE THERAPEUTIC INDEX A concept which has been useful in pharmacological research and which has been applied in ex- Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. GOLDIN et al.--Preclinical Screening and Evaluation p e r i m e n t a l cancer c h e m o t h e r a p y is t h a t of the " t h e r a p e u t i c index." T h i s index is one which takes account of the fact t h a t the action of an a g e n t has a d u a l nature. T h e r e are effects b o t h on t h e disease process for which the a g e n t is being applied a n d on the diseased host. W h e n a p p r o p r i a t e l y e m p l o y e d , the t h e r a p e u t i c index is a p a r a m e t e r , v a l u a b l e in characterizing the action of a t h e r a p e u t i c agent. I n general, the t h e r a p e u t i c index m a y be used to rate a n t i t u m o r agents w i t h respect to the m a r g i n of safety in their use. T h e higher the t h e r a p e u t i c index, the greater the m a r g i n of safety in the a c h i e v e m e n t of a specified a n t i t u m o r effect. Specifically, the t h e r a p e u t i c index is the ratio of the dosage level of a n agent causing a defined degree of d a m a g e in the host to the dosage causing a defined effect on the disease process. D a m a g e to the host m a y be in t e r m s of lethality, weight loss, or other toxic effects of the agent, the defined degree of such effect o r d i n a r i l y being a t some minim a l level. R e l a t i v e to the disease process, the defined effect m a y be some specified reduction in p o p u l a t i o n of the disease-causing e n t i t y below either its initial value or the final value for corresponding u n t r e a t e d controls; a l t e r n a t i v e l y , the effect on the disease process m a y be in t e r m s of a n objectively m e a s u r e d degree of i m p r o v e m e n t in the host, or a certain percentage of cures, or a certain survival time increase. W i t h e x p e r i m e n t a l t u m o r systems, the t h e r a p e u t i c index m a y be t a k e n as the ratio of a specified host toxicity to a specified a n t i t u m o r effect (56, 57, 7~, 84). F o r purposes of the current discussion, it m a y be defined as LD~0/EDg0, where the LD10 is the dosage lethal to 10 per cent of the test a n i m a l s a n d the EDg0 is the dosage yielding a 90 per cent reduction in tumor weight below u n t r e a t e d controls. T h e m e d i c a l a d v a n t a g e s of a h i g h t h e r a p e u t i c index in some situations can readily be seen. Suppose in a clinical situation t h e index were defined in t e r m s of the ratio of dose j u s t b a r e l y p r o d u c i n g toxic m a n i f e s t a t i o n s in the p a t i e n t to the dose giving some large percentage of cures. W h e r e t h e ratio is high, a t r e a t m e n t dosage l e v d can be selected somewhere between the effective dose a n d the tolerated dose so as to provide a wide m a r g i n of safety. T h e r e is e n o u g h l a t i t u d e to ensure little chance of a p a t i e n t ' s being either overtreated or ineffectively treated. An a g e n t with a high index is thus ideal for routine medical use with only fairly general instructions to clinicians as t o app r o p r i a t e t r e a t m e n t levels. T h e lower m a r g i n of safety which would o b t a i n when the t h e r a p e u t i c index is low could restrict the use of a n a g e n t to eases where conditions can be carefully controlled w i t h respect to the characteristics b o t h of the pat i e n t a n d the drug. I t m i g h t be too optimistic to 1841 expect a clear-cut t h e r a p e u t i c a d v a n t a g e in m a n w i t h a d r u g which has only a m o d e r a t e increase in t h e r a p e u t i c index in a n a n i m a l t u m o r system. R e t u r n now to the t h e r a p e u t i c index defined i --i--" -- i 35- '1" [ 1 i I t CARCINOMA 755 EXP. I-FOOD RESTRICTION FROM DAY 4 {)-6M& FOOD OFFERED ,~ 25PER DAY PER A /~_.z(r MOUSE /~%z~ .... uS //~N.//\'~--,,._._._._._._.~.(2.2) 2030 tD Z -I, l { I 15 I0 g ~o(.m (C)=AD LIBITUM CONTROLS iV;~'~(o.-.~) ' I=MEDIAN SURVIVAL TIME 5L 3 ~ - I -+--, I F i r t t I EXP. 2-FOOD RESTRICTION FROM DAY I0 w 30- ~ 25 20 < rY uJ ' i ~ 15 I0- /~ ---~(2.a) -40 3) q (o.8) iJ 50 0 -I, 8 16 24 32 i 40 DAYS POST TUMOR INOCULATION Cm~T &--Effects of food restriction oi1 Carcinoma 755.Results are shown in the same way here for food restriction effects as they are for drug treatment effects in Charts ~a, ~b, 3a, and 8b. For explanation of what is shown, see legend for Charts ~a and ~b, first paragraph. The top panel shows how food restriction can mimic the effects of a compound which, though inhibitory to the tumor, is ineffective in eliciting important survival time increases. Extreme food restriction, like overdosage with some drugs, leads to more pronounced tumor inhibition but survival time is reduced. For the experiment of the bottom panel, the initiation of food restriction was delayed until day 10. In this experiment, food restriction does lead to an extension in survival time (untreated controls died sooner in this experiment than in the first). As with overdosages of an effective agent, the more extreme tmnor inhibitions with still further food restriction caused earlier death of the animals. That food restriction can produce some degree of extension in survival time does suggest that agents to be judged effective should be required to yield still greater extensions when working with this experimental tumor system. The phenomenon of food restriction being more effective: when initiated late than early parallels effects observed with some chemical agents. If treatment with a tumor-inhlbitory agent having cumulative toxicity for the host is initiated early, it will kill the host before the tumor does; with delayed initiation of treatment, drug lethality occurs after the normal time of death from tumor. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. 134s Cancer Research above as the LD~0/EDg0 in an experimental tumor system. Clearly, it is not sufficient to inhibit a tumor only 90 per cent; it may still be growing or m a y resume growth and destroy the host. Suppose one were willing to tolerate 10 per cent drug lethality for the host. Then, where the index is high, a large relative dosage increase over the EDg0 can be effected without encountering undue lethality, while for a low index only a small increase can be tolerated. The increased effect on the tumor depends, however, not only on the dosage increase but also on the nature of the dosage-response curve for the tumor. Where there is parallelism of these curves for the two agents, ordinarily the larger dosage increase permitted with the high therapeutic index would result in greater antitumor effect. Such parallelism may not, however, exist. I t is possible that the agent with the lower index may nevertheless display a so much steeper antitumor dosage response curve as to prove more efficacious at the tolerated level. The agent with the greater index may have so shallow a curve as to be little more effective against the tumor at the LD10 than at the EDg0. The therapeutic index may not be too discriminating if it is employed with weak or highly sensitive tumor systems. If the tumor system is extremely sensitive, any number of drugs having different therapeutic indices could effect complete inhibition of tumor growth. In such circumstances, the assay system would, in essence, determine the degree of safety with which any number of drugs may effect total inhibition of tumor growth. A common practice in determining the therapeutic indexes of compounds is to obtain the host toxicity data in normal mice, with the tumor mice exclusively used to determine degree of a n t i m m o r inhibition. An additional element of error is involved in this procedure, since there may be a lack of correspondence of the dose-mortality curve for the normal host and the tumorous host. In general, the tumorous host may tolerate less of the drug. Additional studies would be required to compare drug toxicity in the normal and tumorous host. Even if the drugs are so inhibitory to tumor growth t h a t the dose range for antitumor effect is much below the dose range for toxicity, the most efficient practice could be to determine the entire dose-response curves for tumor and host in the same population of tumorous animals. If it is considered t h a t this dose range is too great, it might be useful in order to bring the antihost and antitumor dose ranges closer, to alter the test system so t h a t the antitumor effect is more difficult to obtain. Vol. ~1, N o v e m b e r 1961 THE SPECIFICITY INDEX While intended primarily as a measure of drug safety the therapeutic index may, under appropriate circumstances, be employed to compare the therapeutic potencies of various drugs. This is true when the tumor-dose response curves and also the host-dose response curves for the various agents are parallel. This m a y occur if the various agents being compared have related structures and thereby display similar activities. Care, however, should be taken in comparing therapeutic indices, particularly for unrelated structures. In general, when the agents do not have similar activities, the therapeutic index fails to provide an answer to the question, " F o r equivalent cost in toxicity or equivalent risk for the host, which drug is the more effective antitumor agent?" An alternative measure, the specificity index, has been employed (~27, 33-39) as a measure of therapeutic efficacy. In broadest terms, it is (lcfined as the antitumor effect at a specified toxicity risk. The question is asked, " F o r equal host toxicity, which drug gives greater inhibition of tumor growth, or greater increase in survival time, or greater percentage cure, etc.?" Other paralneters of host effect and tumor effect could be employed. The host effect could be body weight change, food intake, hematopoietic alteration, or a cytological or biochemical response, etc. The tumor effect could be change in cell number, level of enzyme, cytologic alteration including mitotic aberration, etc. In principle, there is not only a specificity index for an agent but also a specificity curve. Suppose an agent has been administered to the tumorous host under conditions which permit ascertaining separate dose response curves for both tumor and host. A specificity curve can, in turn, be determined from these curves by relating the antitumor effect of drug treatment to the corresponding effect on the host. Drugs which have distinct response curves for tumor and host may yet have identical specificity curves. Chart 6 illustrates this concept. For each of two drugs, A and B, the panels of the figure illustrate the tumor response curve, the host response curve, and the specificity curve. In the illustration, drug A, which has the lower response curve both with respect to the tumor and the host, has, nevertheless, the higher specificity curve. For simplicity, in the illustration, the response curves are shown as parallel straight lines; this is not a general requirement for obtaining or comparing specificity curves. The methodology may be employed to rate drugs with respect to specificity, or to evaluate Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. GOLDIN et al.--Preclinical Screening and Evaluation regimens of therapy. This type of evaluation has been made with individual drugs (38, 39) and combinations of drugs (~7, 3~-34, 37, 39) used in treatment. The combinations of drugs have been employed in studies of synergism (~7, 3~, 39) and antagonism (~7, 3~-34, 37). In the above types of assay the question may arise as to how to determine dose-response curves for host and for tumor in the same host. This has been accomplished in one type of experiment in which host. mortality was used as the toxicity index and survival time of iniee not succumbing to host toxicity as the tumor index, by designing the experimental test system to provide a temporal separation between the two parameters. In this system, animals succumbed to drug dose toxicity prior to the time that untreated controls suecumbed to tumor. R E L A T I O N S H I P B E T W E E N T t t E TIIERAP E U T I C I N D E X AND TIIE SPECIFICITY INDEX If the therapeutic specificity of an antitumor agent is defined in terms of its antineoplastic effect at a defined level of tolerance for the host (specificity index), it is seen that there is no necessary relationship between a drug's therapeutic specificity and its therapeutic index. 1 The therapeutic index has to do not so much with the efficacy of an agent, but rather with the care needed to be exercised in its employment. The importance of both the efficacy of an agent and the degree of safety in its use is evident. Since, in clinical cancer situations the utmost of care is exercised in the use of a drug, additional attention should be given to therapeutic specificity as an important basis for ranking candidate agents. The concepts of the therapeutic index and therapeutic specificity have certain parallels in the assay procedures involving maximum survival time increases. As described above, the assay is directed toward ascertaining the maximum survival time which can be obtained with a drug on a particular schedule. This maximum can be thought of as corresponding to the drug's therapeutic specificity. Use of dosage levels either above or below the optimal will result in shorter survival times. Assume there is some survival time which, though below the maximum, is nevertheless desirably high. It should be borne in mind that, as with the therapeutic index, nonparallelism of dose-response curves m a y also posc a problem for the estimation of the specificity index. Alteration of the specificity index at different toxicity levels with nonparallel dose response curves for t u m o r and host m a y create additional difficulties in attempts at correlating therapeutic index and specificity index. 1343 There should then be two dosage levels yielding this survival time; one will be above, the other below, the optimal dosage. The ratio of these two dosages, which corresponds to the therapeutic index, indicates how much latitude there is for varying the dosage without encountering undesirably low survival times. This index has been employed by Creech et al. (13) and can provide an important ancillary index to determination of the maximum survival time in the rating of chemotherapeutic agents. The various concepts discussed in this section are illustrated graphically in Charts 7 and 8. TIIE TUMOR CIIALLENGE Any experimental measure of the effect of an antineoplastic agent is necessarily specific to the circumstances surrounding its determination. Effectiveness will vary with the particular neoplasm 8,00] -f- i001 ~ oI, O so l0 20 410 I~ Olll0 DOSAGE SCALE r I00 o ~ 5O I,I Lt- 210 410 DOSAGE SCALE I so % ~o ,oo EFFECT ON HOST C~IART & - - S c h e m a t i c representation of the use of the specificity index to determine tile relative effectiveness of two drugs, A and B. The upper panels illustrate dose response curves for the t u m o r and the host. T h e vertical scaling is not in defined units and should be interpreted to be the appropriate scale for a n y appropriate parameters of t u m o r and host response employed (increasing numbers indicate increased response). The dosage scale was chosen arbitrarily. It should be noted that, for equal dosage, the a n t i t u m o r effect of drug B is greater than that of drug A. The deleterious effect of drug B for the host is also greater than t h a t of drug A. The lower panel illustrates, for each drug, the antitumor effect as a function of the effect on the host. From these specificity curves, it can be seen t h a t for a n y given effect on tile host, drug A produces a greater a n t i t u m o r effect than drug B. For simplicity, the dose response curves for drugs A and B are showu as being parallel. Nonparallelism between the dose response curves for the t u m o r or between the dose response curves for the host could result in nonparallelism between the specificity curves for the two drugs. This would complicate their evaluation to the extent t h a t the relative efficacy of the drugs would vary with the level of effect on the host chosen for their comparison. Examples of the use of the specificity index for determining the relative merits of various therapies m a y be seen in Charts 10 and 11. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. Z HIGH SPECIFICITY A ANTI-TUMOREFFECTI00 AT DESIGNATED TOXICITYFOR HOST B I00 < F--~.) ANTI-TUMOR CURVE//J r, u.. "' 80 ~,- 60 -THERAPEUTI - HIGH 4~ o~ LOW THERAPEUTIC/~ INDEX 'b9-o t6 "2 / I / OR TU OR TUMO MO~,,R,-" / 0 C ts.l V') ..aO >- 60 a: 40 < 20 LOW. SPECIFICITY D/ ANTI-TUMOR EFFECT40 AT DESIGNATED TOXICITY FOR H O S T / / HIGH THERAPEUTIC INDEX LOW THERAPEUTIC INDEX// <=80 Q) I--rfl n.-" l "~'DESIGNATED, /' 9a~ O ta_ I - - r'r < .HOST I TOXtCtTy 7 12) 3 2 0 . nO ,/ ~ _ 3~o 2 / L/ ~/ / I0 20 40 80 I 160 320 640 1280 20 40 80 DOSAGE SCALE ~ ~ , SURVIVAL / ' I I i , 6O - \ 60 - , ' / / l I I I -i /I j /I NARROW - \ (DOUBLED \ I A I00 DAYS WIDE EFFECTIVE I SURVIVAL CONTROLS a: 80 - I B / X, a',ME) ,'--, 40 - ./~nnt JpJ ~n~l ~WvAIN,, 32o = ,6 ..... . . . . . . J % 0 , MAXIMUM SURVIVAL TIME CrIME ___2O F- 160 320 6401280 HIGH EFFICACY I00 o'3 I ] Panel A illustrates an agent with a high therapeutic index (thc ratio of the dose yielding a designated level of toxicity to the dose yielding a designated antitumor effect is large) and a high st)eciti(tity (there is a strong antitumor effect at a dosage level yielding a designated toxic effect for the host). In panel B it can be seen that the same high specificity can be obtained even when the therapeutic index is low, whereas panel C illustrates how tlle high therapeutic index can be coupled with a low st)ecificity. Finally, panel D shows an association ()f low theral)eutic index with low specificity. r A 80 I~ / / / CIIART 7.--Schematic representation of various possible relationships between the antitumor speciticity of an agent and its therapeutic index. Each of tile four panels of tim chart shows a hypothetical autitumor dose response curve and a hypothetical host toxicity curve for an agent. For simplicity, these are shown as straight lines. The vertical scaling is not in defined units and should be interpreted to be tlm appropriate scale for antitumor effect and host effect as the case m a y be. Antitumor effects and effects on the host are generally in noneomparal)le units making the concept of parallelism between their dose response curves inappropriate. , ] I I ~ G E l ~^ I -! :, _~ i ,~O=z 80 \ ~ _ ~ L C WIDE EFFECTIVE I I L I I I I I I J LOW EFFICACY D MAXIMUM SURVIVAL TIME -I 50 DAYS NARROW EFFECTIVE DOSE RANGE, z-~bg-o ~m I _ DOSE RANGE, 40 20 0 I0 CON TROL S "~ I ; ( 20 40 80 i I I :I 160 320 640 1280 r 20 : ' 40 80 ! I I i 160 320 640 1280 DOSAGE SCALE CHART 8.--Schematic representation of various possible relationships between the therapeuti(' efficacy of an agent and the width of its effective dose range. These relationships are intended to parallel those between antitumor spccificity and therapeutic index as shown in Chart 7. Each of thc four panels of the chart shows a hypothctical survival time dose-response curve. Characteristically, the survival time of treated tumorbearing animals increases with dosagc up to a I)oint; increased dosage beyond some optimal lcvel results in reductions rather than increases in survival time. The therapeutic ctficacy of an agent is defined as the survival time elicited by its optimal dos- age. Arbitrarily here, the width of tile effective close range is defined as the ratio of the highest dose yiehting a twofold or better increase in survival time over untreated controls to the lowest dose doing so. I t can be seen from the various panels that agcnts with high efficacy (panels A and B) m a y be effective over either a wide or narrow ctt'cctive dose range; also agents having a wide effective dose range (panels .4 and C) m a y or m a y not be especially efficacious. These parallel the possible situations existing between the antitumor specificity of a drug and its therapeutic index as shown in Chart 7. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. GOLDIN el al.---Preclinical Screening and Evaluation 11345 The work of this laboratory has been characemployed and its source, and with the particular host employed and its age, sex, or weight. Varying terized by the use of increasing levels of t u m o r the route of t u m o r inoculation or drug t r e a t m e n t challenge as more effective agents have been idenm a y result in changed measure of effect (45, 100). tiffed (44). Thus, when it became apparent t h a t Altered schedules of drug therapy m a y increase or complete inhibition of mouse leukemia LI~10 diminish effectiveness (85, 87, 40, 97). Changes in could readily be achieved with early drug treatdefinition also can change one's measure of effec- ment, provided the t u m o r inoculum was barely tiveness. Defining the effective a n t i t u m o r dose as large enough to ensure 100 per cent takes, the chalthe one yielding 50 per cent t u m o r weight loss in lenge was made more difficult. T h e inoculum was 5 days will give different measures of the therapeu- raised to one about 100 times t h a t giving 100 per tic index than defining it as the dose yielding 90 cent takes in the t u m o r titration. Effectiveness in per cent inhibition in 7 days. Alternative measures such a system now became the criterion for rating can flow from defining the effective dose in terms agents. An increasing measure of success against this of effect on t u m o r diameter or on survival time; similar changes can occur from changes in defini- massive inoculum in turn led to the employment tion of the tolerated dose, as, for example, the of still more extensive t u m o r challenges. I t was dose yielding not more than o0 per cent weight recognized that, while appropriate therapy did loss or the dose yielding not more than 10 per cent lead to "cure," even though the inoculmn emlethality. ployed was massive, nevertheless, t r e a t m e n t had Changes in measure of drug effectiveness such been initiated while the t m n o r was still not y e t as the last, resulting from altered definitions, are palpable. This did not approximate very well the only of academic interest. Others m a y be of impor- clinical situation. T h e further increase ill the tut a n t theoretical interest. I t m a y be worth while to mor challenge made at this point was one in which learn why a particular drug is effective with a cer- test animals were not subjected to drug therapy tain host, or more effective when the host is older, until tumors were palpable at the site of inoculaetc. Other causes of altered drug effectiveness m a y tion and the disease was systemic. With this challenge amethopterin was more efbe of important practical interest. If variation in method of use of an agent, as by altered schedules fective than the various known antileukemic of therapy, can make the drug appear to be more agents. I t produced several weeks' extension in or less effective, how most effectively to use the survival time, even though t r e a t m e n t was withagent is worth learning. Although somewhat rea- h e l d until only 5~ or :3 clays before death would sonable a t t e m p t s can be made at determining es- otherwise have occurred. This provided a yardpecially effective schedules of therapy in the lab- stick for comparing the effectiveness of still other oratory, in the clinic this remains one of the less agents. :Finally, use of the assay procedure employwell resolved problems. ing delayed initiation of t r e a t m e n t led to the findAn important factor affecting measures of drug ing t h a t certain halogenated analogs of folic acid effect which has been referred to in the foregoing could be used routinely to obtain a high percentis t h a t of the t u m o r challenge employed. This fac- a g e of cures. No little p a r t in the cure of experiwhich only a few years tor is germane to the design of a program for iso- mental leukemia LIs lating especially effective agents, whether con- ago was thought to be among the more virulent of ducted on a mass screening or on an individual the laboratory neoplasms, w a s played by the use of a criterion of efficacy which emphasized the laboratory basis. Ordinarily, the more extreme the neoplastic therapeutic results achieved rather t h a n the (Rose challenge employed, the more difficult it will be to level employed. On an equimolar basis, amethopfind efficacious agents. The use of rather weak terin exerts greater antileukemic effect than 3',5'challenges has some justification in the early dichloroamethopterin. However, the ha]ogenated stages of a chemotherapeutic research program. I t analog is considerably less toxic for the host. This m a y serve at least to demonstrate t h a t the t u m o r permitted the use of relatively massive doses of the is susceptible to t r e a t m e n t and m a y provide a halogenated analog, resulting in extensive antistarting point for further improvement. Con- leukemic effect unaccompanied by host toxicity tinued use of too low a t u m o r challenge can, how- (51, 4~, 99). ever, produce a misleading number of laboratory I t m a y be noted t h a t use of a lesser t u m o r chalcancer "cures." With weak challenges it is difficult lenge may, under certain circumstances, provide a to differentiate between agents having specific or reasonable basis for rating candidate agents. When nonspeeifie a n t i t u m o r effects, or between agents the challenge is limited, one can limit the extent having varying degrees of specific effects. to which the test compound is used. Thus, when Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. 1346 drugs were t e s t e d in this l a b o r a t o r y a g a i n s t t h e more m a s s i v e t u m o r i n o c u l u m , b u t w i t h t r e a t m e n t given early, such t r e a t m e n t was given on o n l y a single or on o n l y a l i m i t e d n u m b e r of days. As ind i c a t e d above, this device p e r m i t t e d a s e p a r a t i o n of t h e l e t h a l effects a n d t h e a n t i t u m o r effects of such t r e a t m e n t . T h e agents could t h e n be r a t e d on the basis of t h e specificity indices (38). T h e v a r i o u s systems e m p l o y e d in this laborat o r y h a v e been useful n o t o n l y for r a t i n g c a n d i d a t e l I l I I 1 i I TREATED (LOW INOC~LUM _J I00 <~ > ! )~ 80- ~jL____~CONTROL 1~ / .~ I~(HIGH INOCULUM) ~o uJ II~CONTROL !-~_~WLNOCULUM) 40 ! ~X_ a_ 2o o Vol. s Cancer Research I o I0 I 20 I 40 I\ 50 I 60 I I 70 I 80 1961 T h e value of t u m o r i n h i b i t i o n , per se, as a measure of effectiveness is limited, e v e n w h e n t h e restriction is m a d e t h a t such i n h i b i t i o n be n o t acc o m p a n i e d b y u n t o w a r d toxic effects for t h e host. I n concept, t h e r e are a v a r i e t y of m o d e s of a c t i o n b y w h i c h an a g e n t , n a y p r o v i d e a n effective t h e r a p y w i t h o u t its being reflected in a h i g h degree of t u m o r i n h i b i t i o n a t some specified time. T h e r e is no necessary c o r r e l a t i o n b e t w e e n t u m o r size a n d s u r v i v a l time. E x a m p l e s h a v e been given where i n h i b i t i o n of t u m o r g r o w t h was a c c o m p a n i e d b y e i t h e r a n increase, or no increase, or a decrease in s u r v i v a l time. T h e e x t e n t to which d r u g t r e a t m e n t increases t h e s u r v i v a l t i m e of t u m o r - b e a r i n g a n i m a l s is suggested as a m e a s u r e of d r u g effectiveness which gives weight to b o t h t h e a n t i t u m o r effects of t r e a t m e n t a n d its toxic effects for t h e host, a n d which should be a p p r o p r i a [e w h a t e v e r t h e m o d e of t h e r a p e u t i c action. T h e s u r v i v a l t i m e m e a s u r e m e n t re- 1 TREATED ~[-~H-FGH iNOCULUM) ' 30 November i I I I 1 I 28 90 DAYS CHART 9.--Illustration of how Leukemia LI~10 is less responsive to treatment with amethopterin when the tumor inoculum level is high than when it is low. This chart is taken from Goldin et al. (44). The chart shows for each treatment group and its corresponding control the percentage of ten mice surviving to any specified day following tumor implantation. Both the low tumor inoculum (14,000 cells per mouse) and the high tumor inoculum (500,000 cells per mouse) resulted in 100 per cent tumor takes and subsequent death, with only a moderate displacement between the two control survival curves. With a high tumor inoculum, treatment with amethopterin at a level of ~4 mg/kg on days 8 and 7 post-tumor inoculation produced a substantial extension in survival time over untreated controls, but ultimately all mice succumbed to leukemic growth. With the low tumor inoculum 80 per cent of the treated mice survived for 90 days. agents, b u t also for e l u c i d a t i n g t h e effects of exp e r i m e n t a l modifications. Successive kinds of assay p e r f o r m e d in this l a b o r a t o r y e m p l o y i n g progressively m o r e severe challenges are i l l u s t r a t e d in C h a r t s 9-lB. SUMMARY AND CONCLUSIONS Various aspects of t h e p h i l o s o p h y a n d c o n d u c t of a n t i t u m o r drug screening a n d e v a l u a t i o n programs are reviewed a n d i l l u s t r a t e d in t h e p r e s e n t report. T h e yield of screening a n d e v a l u a t i o n p r o g r a m s is influenced b y t h e e x p e r i m e n t a l procedures employed, t h e e n d - p o i n t s observed in m e a s u r i n g d r u g effect, a n d t h e r a t i n g procedure b y w h i c h drug effectiveness is e v a l u a t e d . AMETHOPTERIN 26 i: 24 o _~ I- ~.J ~ / A M NOPTERIN i 2220 IE ',' 18- CONTROLS 16- 14 I I I I I I I 2 3, 4 5 6 7 8 CALCULATED PROBIT OF TOXIC MORTALITY CtIART 10.--Illustration of the survival time vs. probit of toxic mortality procedure for comparing the antileukemic (LI~10) effectiveness of agents. The chart is from Goldin et al. (38), and the basic data are available there. This chart shows only the lines fitted to the basic data. The line for amethopterin being higher than that for aminopterin is interpreted as meaning that, for a given cost in lethality for the host, amethopterin yields the greater antitumor effect. The antitumor effect is evidenced by the average survival time of mice not dying early of drug toxicity, but succumbing later to their tumors. On this basis, amethopterin is the superior agent of the two. As explained in the reference (38), employment of this assay procedure is based on a temporal separation of drug deaths from tumor deaths. Probit procedures (10) are used to ascertain the lethality of any particular level of drug treatment. In turn, least-squares regression procedures are used to relate the data on times of tumor deaths to the calculated probits of toxic mortality. Drugs were administered in a single dose 3 days post-tumor inoculation. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. TREATMENT OF ADVANCED LEUKEMIA(LI210) I I" ' ' I ' I ' 1 ' I ' I ' I r 30 I I I I I I I - t21 I 26_ .,... DAY OF AMINOPTERIN TREATMENT 20 - Dv *- o~ Dichloro-0mmopterln \3'5' _ 223 i-~ J 20-- ~g ,s- / dl w f6 ut ~ ,> ~" P2, t DAY | r~Treotment 5F / / ( N O NO APPARENT APPARENT R REGRESSIONAGAINST ~-"'J ----CONTRoL:AY - 9 0/I 14 ~z 0 /AMINOPTERIN 0.06 I t I I L ! ! from , t ~ I 0.16 t i [ I I , :3.35 9,:30 DOSE , I 25.9 , 1 72 0 (Mg./Kg.) CHART 12.--Illustration of the maximum survival time procedure for comparing the antileukemic (L1210) efflcacies of agents. This experiment is reported in Goldin et al. (4~). These results are based on an experiment in which tumorbearing mice were treated daily beginning on day 8 with graded amounts of drug. While untreated controls died shortly thereafter, all the agents shown yielded clear increases in the median survival time at their optimal doses. The maximum survival time elicited with amethopterin exceeded that for aminopterin, but a higher dosage level was required. This was in confirmation of the results for this drug comparison as shown in Chart 10. With 3',5'-diehloroaminopterin, the optimal dosage was not clearly established, but at its lowest dose this drug did give results comparable to those for amethopterin. T R E A T M E N T OF ADVANCED L E U K E M I A t ~ 9 CHART l l.--Illustration of the lesser susceptibility of the advanced tumor to single treatment with drug. The chart is from Goldin et aI. (35), and the basic data are available there. This chart illustrates how the procedure exemplified in Chart 10 was employed to ascertain the susceptibility of L1210 to treatment with aminopterin when administered as a single dose at various times following tumor implantation. The leukemia was found to be more susceptible on day 3 than on either day 1 or day 6. When treatment was delayed until day 9, no important increases in survival time were noted and there was no evidence of progressive increase in survival time with increasing levels of aminopterin. [-1 I 1.21 DAILY I 2 5 4 5 6 7 8 CALCULATED PROBIT OF TOXIC MORTALITY L 0.4,4 day 8 i I I t L I I I i ( L 1210) (8) t ~ i 1 I '4) (5) I (6) (4) (4) V--- 7o<~ > rr 60- 5~i 40 ) MTX = t ~ 20 W ~0 0|1 CONTROLS ( o ) TREATMENT FROM DAYS7,T0 90 r I i 0.48 0.75 118 I 184 I i I'~1 ! I I t i 171 26.7 41.8 65.5 102 160 DALLY D O S E - M G . / K G . 26.7 4L8 65.5 102 CHART 13.--Illustration of especially efficacious antileukemic (L1~10) compouuds detected using the maximum survival time procedure. The chart is from Goldin et al. (31). In this experiment, amethopterin (MTX) shows its usual high capacity (cf. Chart 12) for extending the survival time of mice bearing advanced leukemia Llgl0. Its dihalogenated forms, 3',5'-dichloroamethopterin (DCM) and 3'-bromo-5'chloroamethopterin (BCM), however, show antileukemic activity of a nature not previously observed. These agents elicit rather extreme increases in median survival time and also yield substantial numbers of 160-day survivors (29) (100-day survivors are shown in parentheses in the figure). With survivors 160 of advanced leukemia established as the reference effect, it m a y become desirable to alter assay procedures to permit detection of agents having the capacity to cure advanced L1210 rather than only to increase survival time. It may be noted that the effective dose levels of the halogenated derivatives are m a n y times that of amethopterin. The increased antileukemic efficacy stems primarily from a sharply reduced toxicity, on a molar basis, for the host, without as extensive a reduction in antileukemic effect. This permits the safe employment of high dose levels with resulting increased therapeutic effects. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. 1348 Cancer fleets the net effect of the drug on the host-tumor relationship. In general, a drug which is capable of eliciting important increases in survival time is likely to inhibit tumor growth. However, a therapeutic effect may be achieved, even when a drug has little or no direct effect on a tumor, i.e. (a) in this laboratory, increase in survival time of mice with leukemia LI~IO has been observed with either progressive growth of the local tumor at the site of inoculation of the leukemic cells, or a decrease in size or disappearance of the local tumor; (b) the drug may have a salutary effect on the host and thereby prolong life. There are other situations in which measurement of local tumor size alone would not adequately dcscribc the therapeutic value of a drug. (a) The drug effect on the tumor may require the mediation of the host. I t may, for example, be necessary for the host to alter the drug to an active form. Such effects, and the toxic limitation of the drug for the host, could bc masked if measurement is limited to local tumor growth. (b) ~Yhere a tumor mctastasizes, measuremcnt of local tumor growth would fail to take into account the infiltration of the disease into vital organs, etc. (c) If tumor size or weight is measured on a specified day and animal weight change is taken into account, this may still not accnrately reflect therapeutic response as adjudged by sm'vival time. For example, the tumor-inhibitory effect or toxicity for the host could occur subsequent to the time of measurement, etc. Thus, a screening system or evaluation program limited to measurement of local tunmr growth m a y miss compounds which produce therapeutic effects without extensive inhibition of tumor growth. In addition, such a screen may give too many false positives. Screening systems employing survival time as the major response with measurement of local tumor growth response as an ancillary index of effect would appear to be highly desirable. Both the tumor response and the survival time of the tumorous animals should be characterized with respect to the influence of nonspecific effects. Regression lines may be established relating host response, tumor response, and survival time response to underfeeding and weight loss of the host. If the system is too sensitive to nonspecific toxicity, steps m a y bc taken to provide a more difficult challenge. This may be accomI)lished by increasing the inoculum level, delaying treatment until the tumor is well established, etc. Survival time measurement lends itself well to precise quantitative evaluation of drug effectiveness. It may be employed in assay systems for the determination of the specificity of action of drugs. Assay systems with survival time used as the primary criterion of drug effectiveness permit the Research Vol. ~1, N o v e m b e r 1961 ordering of compounds in terms of their maximum effectiveness, or in terms of their effectiveness at a given cost in toxicity for the host. The therapeutic index, as a measure of the safety with which a drug may be employed, provides an important index of the action of the drug. Taken in conjunction with the assay of specificity of action it provides a broad characterization of the action of the drug. Preferably, the therapeutic index employed should incorporate survival time as well as tumor size as criteria of drug response. Such assay systems are applicable also to study of combinations of drugs in synergism and antagonism and to determination of the influence of schedules of therapy. The assay system may be employed in pharmacological and bi()chemical studies involving inhibition analysis. The assay systems should remain flexible. 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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research. Preclinical Screening and Evaluation of Agents for the Chemotherapy of Cancer: A Review Abraham Goldin, John M. Venditti and Nathan Mantel Cancer Res 1961;21:1334-1351. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/21/10/1334.citation Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1961 American Association for Cancer Research.