J Cancer Res Clin Oncol (1990) 116:411M24

99239239239 ~esearch 99 9 (~) Springer-Verlag1990

Guest editorial*

Basic principles in preclinical cancer chemotherapy

**

Norbert Brock 1, Jiirg Pohl 1, and Berthold Schneider 2 1 Department of Experimental Cancer Research, ASTA Pharnaa AG, D-4800 Bielefeld 14, Federal Republic of Germany 2 Institute of Biometrics, Medical University, D-3000 Hannover 61, Federal Republic of Germany Received 6 June 1990/Accepted25 June 1990

Summary. Anticancer agents so far available and their mechanisms of action surfer from the problem of their relatively low selectivity. Their insufficient clinical efficacy against the common, slowly growing solid tumors of the lung, gastrointestinal system, kidneys, urinary bladder, and brain remains disappointing. Recently the possibility has been discussed that the limited clinical activity of current anticancer drugs could result from the screening models and methods used in their selection. The initial approach to drug discovery used by the National Cancer Institute, Bethesda, USA (NCI), the greatest oncological research unit in the world, has been empirical large-scale screening in transplantable rodent tumor models. In the past, these preclinical models have been changed periodically in line with retrospecitve analyses of preclinical predictivity for clinical efficacy. Recently, as a new strategy, a "disease-orientated" concept has been developed to screen agents against particular types of human cancer on the basis of the human tumor colonyforming assay in vitro. Each compound should now be tested directly against a spectrum of human tumor lines without passing through a rodent prescreen. Additional assays in vivo may be performed later on. This new screening concept seems to be suitable for identifying the cytotoxicity of new chemical structures and for an evaluation of sensitivity or resistance of the different tumor types. The contrasting concept of "rational drug design" is exemplified by the development of the oxazaphosphorinecytostatics. The basis of this concept was the application of the transport form/active form principle to the antiproliferative nitrogen mustard. Cyclophosphamide,

* The "Journal of Cancer Research and Clinical Oncology" publishes in loose succession "Editorials" and "Guest editorials" on current and/or controversial problems in experimental and clinical oncology. These contributions represent exclusively the personal opinion of the author The Editors '**Dedicated to Professor Dr. Dietrich Schm/ihl on the occasion of his 65th birthday Offprint requests to." N. Brock

the first representative of this group, had already largely reached the given objective. Generalizing conclusions from the different concepts are as follows. 1. Methods and perceptions of general pharmacotherapy must be the principal basis for the development of antitumor compounds. 2. Progress and essential new developments in cancer chemotherapy are based on experiments in intact animais. 3. An important feature of rational drug design is the stepwise or sequential procedure: the design of new drugs is based on the screening results of former drugs to achieve an optimal progress. 4. F o r the analysis of the activity of alkylating agents on rats and mice, the panel of test tumors for screening and pharmacological evaluation must be selected according to a different degree of chemoresistance or chemosensitivity, respectively. It should be aimed at a complete dose/ activity curve with cyclophosphamide as standard at least for the most sensitive tumors of that panel. 5. The therapeutic index, i.e. LD5/CD95, has proved to be a valuable tool for chemotherapeutic usefulness, as has the danger coefficient for the quantification of organotoxic side-effects. These values provide a measure of the therapeutic range and, consequently, of the selectivity of the antitumor activity. Results from a given tumor have proved to be predictive for other tumors and turned out to be relevant also for clinical trials. 6. The different sensitivities o f experimental tumors against alkylating agents is n o t a fundamental property but a quantitative feature. With sub- or even supra-lethal doses it is possible to overcome vitality and transptantability of even the very most resistant tumors. A new product with impressively increased selectivity is consequently expected to achieve remissions in more resistant tumors also. This evaluation system also remains applicable and useful in the context of a "disease-oriented" concept. 7. A profound knowledge of the mechanism of action of cytostatic agents and a deep insight into the metabolic patterns in the host and in tumor cells are the essential basis for a rational augmentation of cytostatic activity.

412 8. Promising leads, identified initially by empirical largescale screening programs, mostly need forther optimization through the rational approach. Thus there is most often an essential and intimate interplay between the rational and large-scale screening strategies. Key words: Anticancer agents - Chemotherapy macology Screening - Drug development

Phar-

Introduction

There are many different ways of discovering new drugs. Originally, drugs were discovered by way of coincidental observations, through knowledge based on past experience in folk medicine or through wide-ranging, untargeted trials with chemical or pharmaceutical products. Since the inception of chemotherapy through Paul Ehrlich, the targeted search for new drugs on the basis of pathophysiological conceptions and biochemical knowhow has advanced into the forefront of drug development. These scientific principles of a goal-oriented search for new drugs also have general validity in the field of cancer chemotherapy, however they suffered for a long time from a great lack ofinsight into the pathophysiological and biochemical fundamentals of the disease of cancer. In this way, just about all new developments and advances in the area of cancer chemotherapy have been acquired through more or less accidental observations and through systematic pharmacological screening in animal experiments, but without an exact knowledge of the biochemical peculiarities of the cancer cell. In spite of this inadequate start, chemotherapy has, in the past few decades, established a secure position for itself in the treatment of cancer in man. By their very nature, the classical methods of surgery and radiation are local or, at best, regional and radical measures and are designed to eradicate all malignant cells from the body. However, by the time they are diagnosed, two-thirds of all malignant tumors have spread beyond local limits. These tumors are generalized, which means that success can only be expected if a therapy with generalized action is applied. This is where the need for chemotherapy arises. Just as in surgery or radiation, the highest aire ofchemotherapy is to cure completely the patient with a cancerous tumor. In light of the irreversibility of the malignant transformation and the cellular heterogeneity of tumors, which increases proportionally to the duration of the disease, the curative goal can only be attained through chemotherapy that is highly selective. But even if a complete cure cannot be reached, suffering can be alleviated and improvement in lire expectancy and quality of life can be achieved through chemotherapy in combination with surgery and radiation (adjuvant therapy), through preoperative treatment (neoadjuvant therapy) and through elimination or lessening of secondary damage (palliative therapy). A particular problem in the setting of chemotherapeutic goals rests in the fact that cancer comprises an extraordinarily complex and diverse group of diseases that share many common biological characteristics col-

lectively defining malignancy. However, specific forms of cancer possess highly distinctive features that defy any global biological groupings. Thus, distinctly different strategies may be necessary for the development of effective antitumor drugs. In a historical context, one of the first pathophysiological principles lay in the fact that the malignant characteristics of rapid growth represent an important attack site for chemical cancer therapy. Cells that are proliferating and have a correspondingly high metabolism are not only much more sensitive to many toxic drugs than dormant cells, but also depend to a particular degree on the appropriate metabolic components, e.g. for the synthesis of nucleic acids in the nucleus and the cytoplasm. The substances preferably used today in cancer chemotherapy and whose development is being pursued in many countries with growing interest in research can be grouped into the following main categories: 1. Alkylating agents electrophiles. 2. Antimetabolites. 3. Antibiotics. 4. Alkaloids. 5. Hormones - antihormones. 6. Enzymes. 7. Other mechanisms. Alkylating agents and electrophilic metals (Fig. 1) attack the DNA directly and modify it through chemical reactions in such a way that replication can no longer take place. Antimetabolites supply false components that are incorporated into newly synthesized DNA or RNA and thereafter further inhibit correct transcription or translation. Several antibiotics intercalate in between the strands of the DNA double helix and prevent further replication. Most alkaloids were initially interpreted as being mitosis inhibitors. Today it is known that at least a few of them are topoisomerase inhibitors. Hormones and hormone derivatives affect tumors with specific hormone receptors on their cell surface which depend on the respective hormones as growth factors. In rare cases, tumor-specific enzymes or enzymes specifically absent in tumors can be used. Today there is a broad range of malignant tumors that can be cured or their growth temporarily suppressed, particularly through a combination of diverse antitumor substances (polychemotherapy). Limitations on the therapeutic use of the various cytostatics are related to the danger that normal tissue with a high proliferation rate, such as bone marrow, lymph glands, intestinal mucosa and gonads of the host organism, can also be damaged. A disappointing fact in cancer drug development is that the clinical drug response of common solid tumors (e.g. of the lungs, the gastrointestinal tract, the kidneys, the urinary tract and the brain) lags far behind that of lymphomas and leukemias. All cytostatically active substance groups and preparations that are used nowadays in the clinical treatment of cancer were discovered by means of transplantable tumor systems in mice and rats. Thus, the possibility is discussed that the limited clinical activity of current anticancer drugs may result from the experimental methods used in their selection. In his recent analysis of anticancer drug development, Muggia concluded that the criteria for identifying new drugs used between 1970 and 1985 in the screening program of the National Cancer Institute (NCI) might have been unsat-

413

Precursors

Asparaginase Anti- folates 6-MP 6-AZU

1

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RNA

J

[

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"

Steroids

"I

1 Proteins

Hydroxyurea Anti- fo/ates 5-FU

Cytarabine

IDeoxyribonucleorides

; ~y

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=

Daunorubicin Adriamycin

1

Nitrosoureas Triazenes

I

Ribosomes

/\

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agents

dichlorides

I

Base analogues

Fig. 1. Clinicallyapplied cancer chemotherapeuticagents and their sites of action (accordingto Connors 1974) isfactory for the detection of successful drugs against common solid human cancers (Muggia 1987).

Screening Methodologically, the first step in developing new substances is drug screening, i.e. deciding whether or n o t a substance exhibits principally the desired efficacy. The basis for this decision rests on investigations in special modal systems (Brock and Schneider 1984). Initially, in vivo models were used almost exclusively for screening of substances active against cancer cells, i.e. standardized animal models in mica and rats. A specific type of a cancerous tumor was induced or inoculated into animals, the trial substance administered and a reduction in the clinical symptoms of the disease measured. The trial substance was then categorized as "positive" or "negative", according to the extent of this reduction. Only the "positive" ones were researched further and, if possible, developed into pharmaceutical agents (Brock and Schneider 1964). In more recent times, in vitro systems (cell cultures) have been available with which, however, only the direct cytostatic action of a substance can be investigated. Since the 1950s the National Cancer Institute (NCI) has been the largest single contributor to all aspects of anticancer drug development. In addition, governmentfunded cancer units and institutes bave been set up in most of the developed countries with limited, but targeted activities and often in close cooperation with the NCI. For decades, the interest on the part of the pharmaceutical industry was relatively small. Only very few

companies had self-contained, in-house anticancer drugdevelopment programs with closely integrated chemistry, screening and pharmacology departments. Despite this, a number of clinically important cytostatics were produced by just these laboratories. At the outset, the goals ofthe NCI and the cancer laboratories of the pharmaceutical industry shared an important common feature: to develop and to define the activity of a new antitumor agent and to make it available to cancer patients as expeditiously as possible. However, one must also recognize the considerable differences in research concepts as well as in the methods that were practiced in order to achieve this goal. These different concepts can be characterized with the catch-words "mass screening" (large-scale screening) and "rational approach" (rational drug design). In the first case, the number of test substances is virtually unlimited, their chemical structure completely different. For this reason, test models bave to be selected that are not dependent on specific substance characteristics but, more importantly, trace as many compounds with cytostatic activity as possible. In the second case, synthesis is based on a concept that is supported by the results of basic research. Optimizing a basic compound through targeted chemical synthesis and the selection of the "best" derivatives also belong in this category.

On the activities of the NCI (large-scale screening) The initial approach to drug discovery used by the NCI has been empirical large-scale screening in transplantable

414 Table 1. Survey on the NCI strategies Compound-oriented experimental screening ( Goldin et al. 1966)

Extension from L1210, $180, Ca755 to 64 mouse tumors 16 rat tumors 20 hamster tumors 9 other in vitro tumors 6 in vitro systems Reduction to 24 specific models with protocols Reduction to six test systems (Geran et al. 1972): L1210 P388 B16 melanoma Lewis lung carcinoma Walker 256 carcinosarcoma KB cell culture in vitro system Compund-oriented large-scale screening

Preclinical tumor panel models (Venditti 1981) Mouse tumors L1210 P388 B16 melanoma Lewis lung carcinoma Colon carcinoma 38 Mammary Ca CDSF1 Human tumor xenografts Mammary, MX-1 Lung, LX-1 Colon, CX-1 DCT-Prescreen and tumor panel models (Venditti et al. 1984) Prescreen: mutine P388 leukemia Tumor panel models as before In vitro-based disease-oriented screen (Boyd et al. 1989)

In vitro human tumor cell line panels (60 90 cell lines) In vivo testing in selected sensitive human tumor cell lines

rodent tumor models (Goldin et al. 1979; Venditti et al. 1984). The preclinical models used in this screening have changed periodically over the years (survey Table 1). These changes in screening methods have resulted from fundamental studies of the biological factors that affect the success of treatment, such as the relationship of tumor cell growth kinetics to drug responsiveness, as well as from retrospective analysis of correlations between clinical and preclinical efficacy. Since 1955, when largescale screening was initiated, more than 400 000 chemical compounds have been screened for antitumor activity. The vast majority of testing has relied on murine leukemias L1210 or P388 as primary screens. The lack of clinical efficacy against the majority of more slowly growing solid human tumors was a serious concern, which led to the adoption of new strategies. Screening against a whole battery of solid murine tumor models was introduced in 1975 and later on was extended by means of xenografts in athymic mice. Because of the considerable expense of in vivo testing on a large number of models in the mouse tumor panel, a filter was added to reduce the number o f c o m p o u n d s screened in the panel by about 90% 95%. The murine P388 lymphocytic leukemia assay was selected as a sensitive and cost-efficient in vivo prescreen. Compounds that showed a modest but reproducible degree of efficacy in killing P388 tumor cells

would then become candidates for testing against the much more expensive solid tumor panel. This panel of transplantable tumors included mouse breast, colon and lung tumors, human tumor xenografts of the saine type and, further, L1210 leukemia and B16 melanoma, which had been of value in the past. However, even this predominantly compound-oriented screening concept did not fulfill the expectations: it was just as impossible to find a natural relationship between the efficacy on xenografts and clinical application in tumor patients, as it was to find one between the efficacy on various transplanted mouse tumors and human tumors or between xenografts and inoculated tumors. Again, the majority of identified antitumor substances were clinically effective against, in descending order, leukemia, melanoma, mammary, lung, and colon tumors. In lung and colon cancer real chemotherapeutic success could be achieved neither in animal experiments nor in humans (Boyd et al. 1989). It was concluded that the disappointing outcome of the screening was inherently biased by two factors: the use of a single leukemia prescreen and by the attempt to represent each main solid tumor site by only one or two models. Consequently, the "disease-oriented" concept was developed as a new strategy in the early 1980s to screen agents against particular types o f cancer. This was based on Hamburger and Salmon's studies on primary cultures of human solid tumors (human tumor colony-forming assay) (Hamburger and Salmon 1977; Salmon et al. 1978). A large number of human tumor cell lines have been established in the past few years, from carcinomas of the lung, colon, ovaries, kidneys and skin (melanomas), malignancies of the central nervous system and leukemias. Each compound should now be tested directly against a spectrum of tumor types without undergoing prescreening. The panel with these human cell lines additionally offers several possibilities for preclinical in vitro therapeutic trials. Since most o f these in vitro lines are highly tumorigenic in nude mice, additional assays could be performed in vivo. The screening facilities at the NCI are currently being expanded to allow for initiation of large-scale drug screening in vitro and for the implementation o f additional disease-oriented cell-line drug screening (Boyd et al. 1989). With the colony assay, it may be possible to find antitumor substances that would be missed by the con~entional in vivo screening system (e.g. by testing on leukemia L1210 or P388). If this assumption can be further proven in the future, the human tumor colony-forming assay may be developed into a valuable tool for antitumor screening. Additionally, the colony assay may have potential as a secondary screening system for identifying the cytotoxicity of new structures and analogs of known drugs and also for indicating which tumor types are sensitive or resistant. The disadvantage of the colony assay when using tumor material that cornes directly from the patient is the fact that only 2 0 % - 3 0 % of solid tumors form colonies in cell culture (Von H o f f 1990) and, furthermore, that the plating efficiency of colony-forming tumors is low and often around 10-4-10 - 87 Fiebig et al. were able to achieve a considerable increase in the cultivation rates by

415 establishing human tumors primarily in the nude mouse (Fiebig et al. 1987). Using this procedure, the rate of in vitro colony-forming tumors could be increased from a initial 20% to over 80%. The ultimate success of this new drug screening concept, however, will depend on the ability of this program to demonstrate a positive correlation between the in vitro response of established tumor cell lines to specific antitumor agents and the in vivo clinical response observed in similar tumor types to the saine agents in a patient's population. Such correlations have hOt yet been available in numbers convincing enough to provide a proof of whether this disease-oriented testing leads to the detection of new principles of action in the treatment of solid tumors.

Rational drug design Basic principle: transport form/active form. The few research laboratories in the pharmaceutical industry that in the 1950s made the courageous decision to place the emphasis of their research on the development of an effective cancer chemotherapy did not find it financially feasible to apply the mass screening method. According to the principle of rational drug design, they concentrated their efforts on the discovery of a specific and promising substance group with the additional goal of improved selectivity. Using ASTA research as a paradigm of other research groups, the goals and methods of this research and development have been described in more detail (Brock 1976, 1977, 1983, 1986). The development of effective drugs with highly selectivity and a sufficient therapeutic range is not only the concern of.experimental cancer research, but a fundamental )roblem of pharmacotherapy as a whole. The experimental pharmacotherapist is familiar with the goals and methods of this field of research (Brock and Schneider 1961, 1966). We realized that there was a good chance of developing effective antitumor substances by applying the basic principles of pharmacotherapeutic development to the special field of cancer chemotherapy, which was something that had not happened to a satisfactory extent up to that time. In arder to achieve success, ideas had to be developed and, by applying strict scientific criteria, tested for their merit in the treatment of the various malignant diseases. As a starting point for our own developmental work, we chose a therapeutic principle that had been suggested by Druckrey and Raabe (1952). According to this principle, a highly reactive, toxic pharmaceutical agent was to be used, not directly but in a chemically disguised, inactive transport form. The non-toxic, inactive transport form had to be structured in such a way that it would be transformed into its active form in the human body, and most preferably in the tumor cell. It should also be mentioned that the development of antimetabolites was approached on the basis of similar ideas and is still being continued today. We chose nitrogen mustard as a drug with general cytotoxic action, which was also able to destroy cancer cells (Arnold et al. 1958). Nitrogen mustard

is a strong antiproliferative substance; it acts very generally on multiplying cells. This has the advantage that all types of cancer cells can be attacked, but also the disadvantage that, owing to the absolute lack of selectivity, vital cells and tissue ofthe host organism are also damaged. Precisely because of these characteristics nitrogen mustard seemed to be particularly suited for application in the principle of transport form/active form (Brock 1958). The first task was to inactivate nitrogen mustard by a appropriate chemical substitution, transferring it into a well-tolerated, inactive, transport form. The biological efficacy of nitrogen mustard compounds is closely related to the reactivity ofthe functional 2-chloroethylamine groups, which again depends on the basicity of their central nitrogen atom. An increase in the reactivity of the chloroethyl groups can be expected with increasing basicity, a decrease with reduced basicity of the nitrogen atom. Substitution of electrophilic groups at the nitrogen atom leads to a reduction in basicity and thereby to the desired decrease in reactivity of the functional groups. The introduction of nucleophilic groups has the opposite effect. The phosphoryl group was selected as the electrophile for physiological reasons and the oxazaphosphorine molecule, which appeared to provide attack sites for the body's own enzymes, was extended. More than 1000 substances were synthesized according to this principle for screening and pharmacological characterization. Trofosfamide and ifosfamide were selected in the 1970s for their pharmacological properties and successfully introduced into clinical practice. In the 1980s, as a result of more profound knowledge of oxazaphosphorine metabolism in warm-blooded animals, the idea emerged of trying to stabilize chemically the unstable primary metabolites and to characterize them pharmacologically and clinically. Mafosfamide is the first promising product of these research efforts. Its indications are associated with purging in autologous bone marrow transplantation and extend all the way in the field of immunomodulation (Fig. 2).

Criteria for selection and methods of testing. Whole-body animal experiments were essential to test the principle concept of metabolizing an inactive transport form into a functioning active form (Brock 1978). In primary

Oxazaphosphorine

R1

1t2

H-

cydophosphamide 4-HydroperoxyCydophosphamide

113

CI-CH2C~ ~NCI-CHzCHz

HOOeO3S-CH2CH2-S-

R1 N--CH

Mafosfamide

O

Ifosfamide

9 N. CI-CH2CH2

CI-CH2CH ~-

H-

Trofosfamide

CI-CHzCH~NCI-CH2CH2

CI-CHzCH U

H-

CI-CH2CH2-

H-

V //\

~.~ /

O--CH 2

H

Sufosfamide

H ~NCH3SO3CH2CH2

Fig.2. Chemical structures of particularly favorable oxazaphosphorines

416

screening, we used the Yoshida ascites sarcoma AH 13 growing in BD II rats and the Walker 256 carcinosarcoma growing in Sprague-Dawley rats. The decisive reason for choosing these two relatively chemosensitive types of tumor was the fact that, under the experimental conditions selected and using cyclophosphamide as a standard, complete dose/response lines can be expected for curative action. The quantitative determination of the various therapeutic and toxic actions of a drug in animal experiments should preferably be made by establishing the corresponding dose/response relationship. Here it is important to have a clear and simple test for each active component, a test whose pathophysiological conditions resemble those ofthe clinical situation as closely as possible. In the past, this was not always taken into consideration in the pharmacological testing of cytostatics. The evaluation of tumor growth inhibition in experimental animals at an arbitrary time after treatment, for example, does not allow for a sufficient assessment ofthe curative efficacy because enhanced growth is often subsequently observed. Clear-cut and fully reproducible results were only obtained when we used the animals's definite cure or the increase in life span as quantitative criteria for the assessment of the drug's antitumor efficacy. For further experiments in animals, various tumor types (solid tumors and leukemias from rat and mouse) were used as test systems. We chose a collection of tumors with increasing chemoresistance for testing alkylating cytostatics. In light of other aspects, selection according to specific biochemical properties or according to the type of organ involved can also be advantageous (Connors and Whisson 1966). Experimental tumors are either transferred by inoculation of the experimental animal (heterologous or homologous transplantation tumors), or they are directly induced in the animal using carcinogenic substances (autochthonous tumors). The latter is possible today with a high specificity in virtually all organs (Druckrey et al. 1967). Chemosensitivity is frequently highest in heterologous transplantation tumors of high-grade malignancy and is lowest in the slower-growing autochthonous tumors. In order to provide for really comparable conditions and thereby a secure basis, screening should begin with relatively chemosentitive transplantation tumors and then, in the case of a possible reaction, extend to more resistant autochthonous tumors. Schm/ihl et al. have developed several of these organ tumors into experimental chemical models (Schmfihl 1976; Habs and Schm~hl 1979; Fiebig and Schm/ihl 1978). Already in the screening stage, the quantitative comparison of the curative and the toxic dose/response curves allows for a specific assessment of selectivity of the antitumor action (Brock and Schneider 1965). We have taken the therapeutic index from the two dose/response regression lines as the parameter, which is defined as the quotient of LD5 to CD95 (Fig. 3). The choice of 5% and 95% values is arbitrary and could just as well be replaced by 10% and 90% or 20% and 80%. Another useful measurement is the D50 index (LD50 : CD50). These indices

Therap.IncL 3:1(27:9) DSO-Ind. 27:I(81: 3) 99

/

95 80 50 20 5 1

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™

LD50

)

DSO-Ind 37

81

Dose

i

2~3

Fig. 3. Determination of the therapeutic index by means of the dose/ action regression lines (Brock and Schneider 1961)

are generally applicable and make it possible to compare directly the efficacy of various substances numerically and in different tumor types. The requirement of approximating the clinical situation not only applies to the characterization of curative and lethal activity, but also to other toxic components: Cytostatics are well known for their numerous organotoxic effects acute as well as chronic - which limit therapeutic use. The acute effects (e.g. nausea, stomatitis, bone marrow depression, alopecia) manifest themselves relatively quickly at the beginning oftherapy; the chronic ones, such as neurotoxicity, cardiotoxicity, pulmotoxicity, hepatotoxicity and gonadal damage, have time constants ofweeks, or months or, in the case of mutagenicity and carcinogenicity, even years. For quantification of the organotoxic action, another parameter, the danger coefficient, has proven to be particularly useful (Brock and Geks 1951). It is based on the relationship between the probabilities of organotoxic and curative action and enumerates the extent of the organotoxic, e.g. leukotoxic or immunosuppressive, risk which accompanies a specific curative dose, for example the CD95 (Fig. 4). If the danger coefficient of a newly developed substance is compared with that of a known standard, the risk of organotropic, toxic complications can be estimated clearly and properly for every curative action. The sensitivity of tumors to alkylating agents shows "individual variations", which is in accordance with the general laws of pharmacology. Itis not of a fundamental nature, but only quantitative. Even in tumors that appear to be completely resistant, an oncocidal effect can be obtained if the drug is supplied to the tumor in adequate concentrations. If cell suspensions of various sensitive or resistant rat tumors are incubated in vitro for 1 h with the directly alkylating chlormethineN-oxide in Ringer's solution, determination of the limit concentration, which

417 counterbalances the tumor's transplantability, will show (Table 2) that the effective concentrations vary considerably ( a t a ratio of 1:100), whereas the full cytostatic effect can be obtained in all types of tumor, even in the highly resistant DScarcinosarcoma. Similar results were seen in vivo in adequate tests on intact animals, where lethal or

99 [%] 9O 75 50 25 D -12,5

il............

Pharmacotherapeutic testing of oxazaphosphorines

1

0,I 0

2,5

5

15

30 [mg/kg]

Dose Danger coefficient DJ2,5[Yo]

Fig. 4. Determination of danger coefficient D for the C D 9 5 by means of the curative (CD) and leukotoxic (ED) regression lines (Brock 1976) Table 2. Mean effective concentrations of chlormethine N-oxide in tumor systems in vitro (1 h at 37~C) according to Druckrey (1961)

Tumors Heterologous

Homologous

supralethal doses abolished the transplantability (vitality) even of the most resistant tumors. The clinical use of all effective compounds is limited by their toxicity. The more c o m p o u n d s with a wider margin of safety were developed, the more the number of tumors amenable to treatment increased (Table 3). Thus it can be concluded that quantitative methods, such as are used for the determination of the margin of safety, allow the transference of the results obtained in a definite type of t u m o r to other t u m o r types and to clinical conditions. This conclusion, however, is in sharp contrast to the assumptions that were discussed earlier as bases for concept variations within the N C I chapter.

Conc. (gg/ml) Walker ascites carcinoma Yoshida ascites sarcoma Jensen sarcoma (homogenate) Tascites sarcoma DS ascites carcinosarcoma C sarcoma (homogenate)

0.15 1.0 3.0 25 25 65

The pharmacological testing of oxazaphosphorines had to adapt itself to the particular character of these compounds. The nitrogen mustard derivatives known to date were either chemically and pharmacologically (e.g. chlormethine, chlormethineN-oxide) highly active or else they were chemically and pharmacologically inactive. The oxazaphosphorines synthesized according to the new concept were, under in vitro conditions, supposed for the most part to be chemically and pharmacologically ineffective; in vivo, however, they were supposed to be therapeutically active and have greatly reduced toxicity. In this way, we began to create the pharmacological and biochemical prerequisites for their testing. The experimental conditions for determining chemical reactivity and biological activity in vitro as well as for the chemotherapeutic evaluation in vivo were developed and standardized. Chemical and biological reactivity

The direct chemical reactivity of the respective compound plays an important role in alkylating cytostatics. There are various assessment methods available: chemically, for example, the test with 4-(4'-nitrobenzyl)pyridine for alkylating activity or the chloride ion release in aqueous buffer solutions for nitrogenmustard deriva-

Table 3. Comparison of chlormethine, chlormethine N-oxide, and cyclophosphamide in seven different tumor strains of the rat according to Druckrey (1963)

Test system

Transplantation

Medium curative doses in therapeutic units (% LD 50) Chlormethine

Yoshida ascites sarcoma Jensen sarcoma Walker carcinoma DENA carcinoma T sarcoma DS carcinoma C sarcoma

Chlormethine N-oxide

Cyclophosphamide

Heterologous

21

4.7

3.0

Heterologous Heterologous Homologous Homologous Homologous Homologous

40 100 87100 87100 87100 87100

4.3 8.2 60 > 100 87100 87100

4.5 4.2 21 50 ~ 100 > 100

418

tives, and, biologically, the incubation test with living tumot cells. Chloride ion release is greatly reduced in oxazaphosphorine derivaties compared to nitrogen mustard itself and to other directly active alkylating agents (Fig. 5). In the nitrobenzylpyridine test, the three oxazaphosphorine compounds exhibit virtually no activity. In the incubation test, the cytotoxically active concentration

is four magnitudes greater than in the directly alkylating substances (Table 4). The test for chemical reactivity and the result of the biological tests unequivocally reveal that the three oxazaphosphorines are virtually inactive in vitro and are thereby true transport forms of nitrogen mustard.

Chemotherapeuticevaluation 2,0

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We evaluated the curative efficacy in vivo on a number of different tumor systems of the rat and mouse (Table 5). This list comprises four rat tumors, selected on the basis of increasing chemoresistance. Furthermore, it includes a monocytic myeloic leukemia L5222 of the rat, which stands out for its special immunological properties (Hilgard et al. 1985; Voegeli et al. 1986; Pohl et al. 1987). Mouse tumors were leukemias L1210 and P388 as well as the B16 melanoma and the Lewis lung carcinoma for interlaboratory comparisons. Extensive analysis of the dose/response relationship was conducted on this spectrum of rat and mouse tumors. Identified promising compounds could then be further evaluated on in vitro tumor panels or on specific animal or human tumor cell lines of different organs, resembling disease-oriented evaluation. Table 6 shows the results of early comparative experiments into the Yoshida ascites sarcoma of the rat receiving a single i.v. administration of cyclophosphamide, chlormethineN-oxide and nor-nitrogen mustard, respec-

Table 4. Cytotoxic concentrations of ifosfamide, cyclophosphamide, Nor-nitrogen mustard, chlormethine N-oxide, and chlormethine in the incubation test with Yoshida ascites cells in vitro

Table 6. Parameters of the curative and lethal effects of cyclophosphamide compared with chlormethine N-oxide and nornitrogen mustard. Single i.v. administration to Yoshida's ascites sarcoma of the rat on the day of inoculation (Brock 1958)

Compound

Agent

LDso (mg/kg)

Cyclophosphamide Chlormethine N-oxide Nor-nitrogen mustard

160 50

Ifosfamide Cyclophosphamide Nor-nitrogen mustard Chlormethine N-oxide Chlormethine

Yoshida ascites sarcoma in vitro (37~C, I h) EC87 (gg/ml) > 1000 > 1000 1.0 0.5 0.1

CDso~ (mg/kg) 4.5 5.4

100

40

LDso (%)

Therapeutic index: LDs/CD95

2.8 10.9

8.5 2.2

40

0.65

Table 5. Test systems for the characterization of new antitumor drugs (ASTA Research Laboratories "

Test system

Host strain

Sp-D heterologous Sp-D heterologous BD-II homologous BD-IX homologous

Tumor inoculum

Parameter of response

Type

Route

AF AF TS SpS B

i.p. i.p. i.m. i.p.

Cured Cured Cured Cured

Yoshida ascites sarcoma AHI3s (sensitive) Yoshida ascites sarcoma AH13r (resistant) DS carcinosarcoma Leukemia 5222

Rat, Rat, Rat, Rat,

Leukemia 1210 Leukemia P388 BI6 melanoma Lewis lung carcinoma

Mouse, Mouse, Mouse, Mouse,

AF AF TS TS

i.p. i.p. s.c. i.v.

Cured animals ILS Cured animals ILS TGI, day 11 Number of lung metastases

KB in vivo

Mouse, NMRI nu+/nu + human xenograft TF

s.c.

TGI, day 21

DBA2 homologous DBA2 homologous C57B1/j6 homologous C57B1/j6 homologous

animals animals ILS animals ILS animals ILS

AF, ascites fluid; TS, tumor suspension; TF, tumor fragment; SpS, spleen suspension; B, blood (heart puncture); ILS, percentage of increase in life span over controls; TGI, percentage of tumor growth inhibition compared with controls a

419 2~

~s r

9 Ifosfarnide I Nor-nitrogen mustard 2 Chlormethine-N-oxide 3 Phenylbutyric mustard d,l-Phenylalanine mustard 5 Mannitol mustard 6 Uracil mustard 7 Chlormethine

9 Cyclophosphomide

20

c~ 16 eB717 eB699 l B 576

12

tB71~ 9 Trofosfomide 2

o

3

0

9 B693 IB617 , ~B70~7

o

5

~0 7

0

I

o

6

, i

o,~

o.e

i

i

t.2

I

!

t.6

I

ZO [vailrnoJ

Fig. 6. Relationship between chemotherapeutic effectiveness and chemical reactivity of some nitrogen mustard oxazaphosphorines compared with various highly reactive nitrogen mustard compounds.

CI" ofter 2g [h]

tively. The curative activity of cyclophosphamide is evidently about as high as that of the reference substance chlormethineN-oxide, but cyclophosphamide is far less toxic than this highly reactive nitrogen mustard derivative. The result is a distinctly larger therapeutic index. There is no obligatory relationship between the toxic and the therapeutic actions: the goal of higher selectivity has indeed been achieved. The graphical illustration (Fig. 6) gives a synopsis of the results of these many years' work. The groups with different properties can be clearly seen in the illustration. Two are near the abscissa axis, that is they only have a very low therapeutic index. These are either highly active and highly toxic direct alkylating agents or nonactive and nontoxic compounds. A third group, with values between 9 and 24, clearly stands out for its high therapeutic index. In vitro, they only have very low reactivity. The oxazaphosphorine cytostatics cyclophosphamide, ifosfamide and trofosfamide belong to this group of active antitumor substances.

Time/action relationship The determination ofthe doses that are used as a basis for the assessment of the therapeutic range (LD5 versus CD95) was performed in the usual "timeless" way with single applications. However, this only appears acceptable if the action is effectively a function of the concentration of the drug; that is, if it is a relatively rapidly reversible "concentration effect". The fact that the cumulative toxicity in just about all cancer drugs antimetabolites, alkaloids and alkylating agents - is very high and often almost completely additive, forces the pharmacologist to investigate the possible dangers of a long-terre treatment more precisely, particularly with regard to practical and clinical application. Generally, the therapeutic value of an antitumor agents is greatest the more rapidly its toxic effect is reversible, and the more its curative effect is

cumulative. According to Druckrey et al., the cumulative properties may be assessed quantitatively by determining that part which is reversible within a certain rime interval, e.g. within 24 h (Druckrey et al. 1963). The cumulation residue C is the complement of the quantity R (reversible part) following the equation R = 1 -- C. The C values can be calculated by comparative determination of the D50 obtained with single administration versus a divided dose of two or more daily fractions. The results of a comparative trial on cyclophosphamide and ifosfamide are shown in Fig. 7. The percentage toxic cumulation residue of ifosfamide after 24 h is about 83% and that of cyclophosphamide is nearly 100%. The curative action, assessed in Yoshidas's ascitic sarcoma, behaves in just the opposite way; for cyclophosphamide the C value is about 45%, whereas for ifosfamide it is about 100%. Thus the curative action ofifosfamide is much more cumulative than that of cyclophosphamide. Based on these findings, a fractionated re-

100

>95

>95

BO

60 -~ 4O 2o 0

Cydophosphamide

Ifosfamide

Fig.7. Cumulative residue of the curative (c~) and toxic (m) effects of cyclophosphamide and ifosfamide 24 h after administration (Brock 1983). Assay design: Sprague-Dawley rats; breeder: Mus Rattus AG Brunnthal; sex: male; weight: 150 190 g. Standard food: Altromin 1324; water ad libitum, specific-pathogen-free conditions; animais per dose: 10; i.v. injection; period of observation: 28 days following the last injection; evaluation: probit method

420 gimen, i.e. distribution ofthe total dose over several days, bas been successfully developed clinically for ifosfamide (Schnitker et a. 1976; Brade et al. 1985). Recently this principle has been extended by administering ifosfamide as a continuous infusion for 1-5 days. This has ruade it possible to reduce toxicity even further, to increase the total dose and to improve the therapeutic results (StuartHarris et al. 1983; Klein et al. 1983).

probability of immunosuppressive effects accompanying a curative dose (CD84), DII shows the probability oftoxicity of the immunosuppressive dose (Table 8). The figures show that cyclophosphamide and ifosfamide are clearly immunosuppressive. Trofosfamide, on the other hand, has only relatively low immunosuppressive efficacy, a finding that has practical importance for its therapeutic application in maintenance therapy.

Assessment of organotoxic side-effects

Models for preclinical pharmacology

Leukotoxic action. A limiting factor in the therapeutic application of alkylating cytostatics is the leukotoxic actiofl, which expresses itself in bone marrow depression. For this reason, a fundamental prerequisite for progress in cancer chemotherapy is the development of substances with low leukotoxicity, measured quantitatively in terms of the danger coefficient. Table 7 shows the probability of leukotoxicity of curative doses of the three oxazaphosphorine derivatives t o b e considerably smaller than that of chlormethineN-oxide as a type of direct alkylating agents. Table 7 also shows that there are still differences in efficacy even among the oxazaphosphorines.

The clinical oncologist expects the pharmacologist to provide him with reliable indications as to the best possible clinical use of the various antitumor agents. Cyclophosphamide and ifosfamide with their favorable physicochemical and physiological properties, their high chemotherapeutic efficacy and their relatively low toxicity were particularly suited for broad experimental application and thereby also for the development of relevant clinical models. Particularly valuable were the numerous observations and experiences provided by clinical oncology that represent the basis for an ever improving cooperation between experimental and clinical medicine. 1. In the attempt to optimize dosage and time factors for clinical application, Druckrey experimentally developed the massive-dose treatment, which has been successfully translated into clinical practice by both Martini and Gross (Druckrey et al. 1963; Martini et al. 1967; Schmitz and Gross 1967). 2. Cyclophosphamide and ifosfamide played an important role in Goldin and Schabel's development of a rational polychemotherapy (Goldin et al. 1974; Schabel 1974). 3. Methods for determining the cumulative properties of cytostatic agents were developed using cyclophosphamide and ifosfamide (Druckrey et al. 1963; Brock and Schneider 1980). 4. Fundamental experiments with cyclophosphanfide are still very relevant in postoperative (Fig. 8) and preopera-

Immunosuppressive efficacy. The immunosuppressive efficacy, an undesirable side-effect, which accompanies tumor therapy with alkylating agents and other cytostatics, can also be quantitatively assessed in experiments. It is important for clinical use to determine the cancerotoxic (curative) and immunosuppressive action with regard to their dose dependence and to place them in a relationship to toxicity (lethality). This, too, is possible using the danger coefficient (Potel and Brock 1972). DI shows the Table7. Leukotoxic effects of cyclophosphamide, trofosfamide, ifosfamideand chlormethine N-oxide. Correlation betweencurative and leukotoxic effects (Yoshida ascites sarcoma) following single i.v. injection in rats Compound

ED 87 ED5o/CDso DCD84 DCD95 (mg/kg) (%) (%)

Cyclophosphamide Trofosfamide Ifosfamide Chlormethine N-ox.

21.3 17 44 11

4.75 8.1 6.8 2

1.2 1.0 0.6 44

12 5 10 86

Table8. Danger coefficient obtained from the curative, immunosuppressive and lethal regression lines for cyclophosphamide, ifosfamide and trofosfamide Drug

tre vo L

¤

%

50

fo c

Danger coefficient(%) DI ~

Cyclophosphamide 0.90 Ifosfamide 4.0 Trofosfamide < 0.02

DII b < 0.02 0.8 4.50

Probability of immunosuppressive action of the DC8~ b Probability of toxic (lethal) action of the fully immunosuppressive dose a

100

Surgery [hemother. alone

alone

40 50 60 70 mg/kg [ombined surgery ptus chemofherapy

Fig. 8. Surgical removal of DScarcinosarcomas (30 g) without and combined with chemotherapy (cyclophosphamide, single dose). Test: definite cure (according to Druckrey et al. 1958)

421

IDEA 80

The principleof to be usedfor

60

transport form (TF)/active form (AF) Nor-Nitrogen mustard

Procedure: Nor-Nitrogen mustard

L

(AF)

aJ

9E L~0

TF

~J

chemicalinactivation

TF

I

enz~jrnaticalactivation in vivo (tumor oeils) ,

AF

20

REALITY A

B

E

D

Fig.9. Comparison of adjuvant and neo-adjuvant cyclophosphamide chemotherapy with surgery and chemotherapy alone. Shay chloroleucoma (tumor weight at the onset of therapy: i0 g). A, Chemotherapy; B, surgery; C, chemotherapy and surgery; D, chemotherapy 7 days before surgery (tumor weight at day of surgery: 0.3 g) (according to Brock 1959)

Nor-Nitrogen mustard

chemicalinactivation nucleophilicsubstitution

enzyrnaticalactivation TF (ex~race41ularand . . . . . l,ular [. . . . . . ])

TFA

livermicrosomes

+

HSR

"

Pharmacokinetics and active mechanisms Based on proof of the relative selectivity of oxazaphosphorine cytostatics, the task was to find the biochemical and pharmacological reason for this phenomenon and to explain the active mechanism. It was ofparticular interest to determine how a nitrogen mustard compound causes selective damage to a tumor cell, since the alkylating reaction that is the basis of the cytotoxic action has to be considered as relatively non-specific. In joint cooperation with Hohorst, we have experimentally investigated the question of specificity and selectivity of nitrogen mustard alkylating agents, and particularly that of the oxazaphos-

AF

TF

(transl~lrt fom~ oi

'

TFA

toxification ~ , 5 ' - e x o n u c ~ 9 tive (Fig. 9) adjuvant chemotherapy (Druckrey et al. 1958; Brock 1959). Van Putten has just recently pointed out the importance of these animal models for clinical oncology and especially for the development of adjuvant or neoadjuvant chemotherapy (van Putten 1986). 5. The probability of undesirable side-effects is quantified using the danger coefficient. 6. Depending on the cell cycle, the efficacy of a sequential therapy, e.g. with vincristine and ifosfamide or cyclophosphamide, has been proved in animal experiments (Klein et al. 1976) and clinically (Klein et al. 1975; Hartwich et al. 1972; K6rner et al. 1975). 7. This limited selection for the clinical reliability of experimental findings on cyclophosphamide and ifosfamide makes it understandable why cyclophosphamide was recommended by the WHO international conference On Screening Methodology for Antitumor Drugs as a comparative preparation for all new and further developments (Geneva 1974). This work has shown that findings on cyclophosphamide and ifosfamide and their predictive value have greater clinical relevance than on many other cytostatics (Goldin et al. 1966; Brade et al. 1985).

,

alkylation

T F A .,g..........~)

~

SR

(ternporary deactivation)

A- polymerase ' DNA

Fig. 10. Ideas and reality in the development of oxazaphosphorine cytostatics. H S R biological thiol

phorines (Brock and Hohorst 1977). This problem has also attracted great international attention and has been extensively investigated by many researchers (Struck et al. 1971; Sladek 1973; Connors et al. 1974; Hill 1975; Foster et al. 1976; Alarcon !976; Creaven et al. 1976; Norpoth 1976; Takamizawa et al. 1976; Colvin 1982). Figure 10 summarizes the development of the oxazaphosphorine cytostatics and our current understanding of their mechanism of action and compares this to the original concept. The desired objective of enzymatic activation of a transport form to an active form in the target oigan has been achieved, even though this involved a series of different intermediate reactions, and the anticipated increase in cancerotoxic selectivity has been convincingly demonstrated. The understanding of the pharmacokinetics and pharmacodynamics of these drugs has guided the modalities of their therapeutic use and provided the basis for the mode of administration and the dosage, especially in polychemotherapeutic regimes. It also provides insight into the temporal interplay of the compounds, the metabolism and the tumor and host cells. However, as the mechanism of action became clearer, and this is another benefit, new ideas for rational further development continually emerged, so that even now, 30 years after the introduction of cyclophosphamide, understanding is still increasing and new projects are being suggested. The development of stable metabolites is in progress and there are now signs that these may be suitable for specific immunomodulation (Brock et al. 1988).

422 Conclusions

In the last two decades, the chemotherapy of malignant tumors has clearly revealed its possibilities, but also its limitations. Approximately 7% of all malignancies, predominantly leukemias, lymphomas, testicular tumors and some types of sarcomas can be cured by chemotherapeutic treatment alone. Further, in about 20% of all tumors, chemotherapy can be administered to achieve a life-prolonging effect (Schm/ihl 1990). Therefore, the problem of the yet non-responding tumors is one of the main research objectives. In light of the disappointing results of chemotherapy in human solid tumors, the NCI has repeatedly and radically altered its screening program to one that is now largely based on in vitro cytotoxicity testing, e.g. the colony-forming or clonogenic assay. The use of diseaseoriented panels of human tumor cell lines - representative of several of the major human malignancies for large-scale drug screening differs fundamentally from earlier antitumor drug screening methods, which employed in vivo models with a few transplantable tumors, mostly leukemias L1210 or P388. The available data of the new disease-oriented panels of human tumor cell lines support the appropriateness of these methods for the feasibility of large-scale antitumor screening in vitro. The central goal of this disease-oriented screening program for identifying those new antitumor drug candidates not discovered by the previous screening programs, however, has not yet been achieved (Shoemaker et al. 1988). Only clinical testing of new leads will ultimately establish or disprove the validity of the new screen for identifying new drugs active against the common refractory solid human tumors. It is questionable whether or not the human tumor colony formation assay will be a sufficient experimental basis for the discovery of new therapeutics. After all, only the intact animal provides the screening model in which the complete and often complex pharmacology of the test compound find its expression. The counterpart of mass screening, rational drug design, which deals with the development and research of a specific group of substances, bas been explained using ASTA research as an example. The goal was the development of antitumor drugs from the group of alkylating agents with highly selective antitumor action. Fulfilling the goal of transferring the transport form/active form principle to the strong antiproliferative nitrogen mustard had led to the development of cyclophosphamide in 1957. The general toxicity of this oxazaphosphorine compound was greatly reduced while its curative efficacy remained the saine. These superior chemotherapeutic properties found in animal experiments were also confirmed in clinical practice in the years following. Many important generalized conclusions can be drawn from the problems dealt with in this research work - especially with regard to the relevance of screening results for the clinical treatment of human tumors: 1. The development of antitumor substances is a pharmacotherapeutic problem. Methods and knowledge of general pharmacotherapy should be utilized in the developinent of antitumor substances.

2. The significant new developments and advances in cancer therapy are based on experiments in intact animais. In recent years, their possibilities have been considerably increased by widening the tumor spectrum, providing adequate model systems for assessing the drug's antitumor and toxic effects by setting down more rigid criteria and using quantitative test methods, especially dose/action analysis, and by pharmacodynamic and pharmacokinetic studies. 3. In the analysis of the efficacy of alkylating agents in the rat and mouse, the selection of test tumors in screening and pharmacological characterization according their chemoresistance or their chemosensitivity has proven itself. 4. The therapeutic index (LD5:CD95) is the most proven parameter for evaluating chemotherapeutic range, and the danger coefficient for quantifying the organotoxic probability. These parameters represent a measure of selectivity. The therapeutic index is hot simply the numerical ratio of antitumor activity to lethality, but it relates to factors responsible for that ratio including the metabolism, the organotoxicity, the pharmacodynamics and the pharmacokinetics, and it makes it possible to rank potential antitumor agents according to their therapeutic value in each special tumor model. This evaluation system is still applicable and useful in future developments (Double and Bibby 1989). 5. To the saine extent to which compounds that have a broad therapeutic spectrum have been successfully developed within a class of substances, the number of susceptible tumors has also increased and the danger of toxic side-effects has decreased (Table 2). From this it can be concluded that these results can be transferred to clinical conditions. 6. The sensitivity of tumors to alkylating substances shows "individual variations", which correspond to the general principles of pharmacology (Druckrey 1961), and, just like sensitivity to radiation, it is more of a quantitative nature than a fundamental one. Even in apparently completely resistant tumors, cytotoxic activity can be achieved in vitro if the chemotherapeutic agent is applied to the tumor cell in a high enough concentration. These results have been confirmed in the corresponding animal experiments in which, after application of sublethal or supralethal doses, vitality and transplantability could be abolished even in the most resistant tumors. The limits for effective substances are therefore set only by their toxicity. If a new product with a significantly broader therapeutic spectrum becomes available, more resistant tumors would also find their way into the realm of chemotherapeutic susceptibility. In this sense, the evaluation system described here is still valid. 7. A targeted improvement in efficacy of the cancer chemotherapeutics available at this time requires an exact knowledge of their mechanisms of action as well as detailed insight into the metabolic conditions ofthe cells on which the various substances have their effect. In the area of basic research, therefore, strenuous efforts should be ruade to discover the various steps involved in malignant growth. In particular, the metabolic processes involved in proliferation are of potential practical interest for possi-

423 ble d e v e l o p m e n t o f a t a r g e t e d c h e m o t h e r a p e u t i c a t t a c k on the cancer cell. A t present, the d i s c o v e r y o f new classes o f active substances with p o t e n t i a l l y c u r a t i v e c y t o t o x i c efficacy is j u d g e d with scepticism. T h e i n t e n t i o n o f d i s e a s e - o r i e n t e d screening initially aires at d i s c o v e r i n g s u b s t a n c e s w i t h org a n - o r i e n t e d efficacy. I f this c o n c e p t b e a r s fruit, p r o m i s ing leads identified initially t h r o u g h large-scale screening s h o u l d be o p t i m i z e d t h r o u g h the r a t i o n a l a p p r o a c h . Thus, there is v e r y often an essential a n d i n t i m a t e interp l a y b e t w e e n the r a t i o n a l a p p r o a c h a n d the different screening strategies. U l t i m a t e l y , the f u n d a m e n t a l s o f pharrnacotherapeutic characterization and development m u s t also be a p p l i e d for ail new leads, as has been d o n e here with o x a z a p h o s p h o r i n e s , to i m p r o v e selectivity a n d t h e r a p e u t i c efficacy.

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