Chemotherapeutics: A questionable or a promising project

Comp. Immun. Microbiol. infect. Dis. Vol. 9, No. 2/3, pp. 99-119, 1986

Printed in Great Britain

CHEMOTHERAPEUTICS: A QUESTIONABLE PROMISING PROJECT

0147-9571/86 $3.00 + 0.00 Pergamon Journals Ltd

OR A

H. H. SEDLACEK,G. DICKNEITEand H. U. SCHORLEMMER Research Laboratories of Behringwerke AG, D-3550 Marburg, F.R.G.

Abstract--While repository adjuvants are already established drugs for antigen specific immunomodulation, no unspecific active immunomodulator has successfully passed clinical trials in tumor patients in Western countries yet. As this is in striking contrast to the effects seen with unspecific immunomodulators in experimental immunological and tumor test systems, the value of those screening models to predict clinical success may be asked for. To improve the success rate, it is recommended to test compounds for their immunomodulatory effects as broad as possible in ex vivo and in vivo test systems. Subsequently, the prophylactic as well as therapeutic potency of selected immunomodulating drugs should be evaluated in various models of aptitude, such as chronic infection, autoimmune diseases and chronic inflammatory reactions. Those diseases are at least to a certain extent influenced by the immune system, in contrast to the uncertainty in case of tumor diseases. In a battery of chronic infection models we could find that different chemoimmunotherapeutics with very similar immunopharmacological activity behaved quite different. Most compounds were ineffective, a part enhanced chronic diseases and only in a few cases a therapeutic effect could be shown. On the other hand, effectivity in protection against the pathogenicity of subsequently applied, selected microorganisms does not predict the therapeutic potency for the same pathogen. Altogether the data show that activation of cells of the immune system by an immunostimulating drug does not predict its therapeutic potency. Moreover, activation of immune cells may also impair the immune resistance. Key words: Predictivity, test system, chemoimmunotherapeutics

R6sum6--Alors qu'il existe d6jfi un certain nombre d'adjuvants reconnus pour leur activit6 immunostimulante sp6cifique d'antig6ne, aucun immunomodulateur actif, non sp6cifique, n'a pour l'instant satisfait aux essais cliniques chez des canc6reux dans les pays Oceidentaux. Comme ceci contraste profond6ment avec les effets observ6s pour des immunostimulants non sp6cifiques dans des tests immunologiques exp6rimentaux ou des tests humoraux, on peut s'interroger sur la valeur de ces tests de criblage pour pr6dire des succ6s cliniques. Pour augmenter le taux de r6ussite, il est recommand6 de tester les effets immunomodulateurs de ces substances le plus largement possible dans des syst6mes ex vivo et in vivo. Ainsi, le pourvoir th6rapeutique et prophylactique des substances immunomodulatrices retenues devra ~tree 6valu6 dans diff6rents tests d'efficacit6 tels qu'infection chronique, maladies auto-immunes et r6actions inflammatoires chroniques. Ces maladies sont, au moins 5. un certain niveau, influenc6es par le syst6me immunitaire, contrairement 5. l'incertitude qui r6gne dans le cas des affections tumorales. Dans une s6rie de mod61es d'infection chronique, nous avons trouv6 que diff6rents agents immunoth6rapeutiques qui avaient des activiti6s immunopharmacologiques similaires se comportaient tr6s diff6remment. La plupart 6taient inefficaces; certains aggravaient les maladies chroniques et ce n+est que dans de rares c a s q u e l'on put observer un effet th6rapeutique. Par ailleurs, l'efficacit6 dans la protection contre le pouvoir pathog6ne de microorganismes s61ectionn6s et utilis6s au pr6alable ne permet pas de pr6voir le pouvoir th6rapeutique vis-fi-vis du m~me germe. Au total, les r6sultats montrent que l'activation de cellules du syst6me immunitaire par une substance immunostimulante ne permet pas de pr6dire ses capacit6s th6rapeutiques. Bien plus, l'activation de cellules immunitaires peut 6galement conduire 5. un affaiblissement de la r6sistance immunitaire. Mots-clefs: Pr6diction, syst6me de test, chimio-immunoth6rapie

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INTRODUCTION What is an immunomodulator, what characteristics is it to have, in what form, on what cells and for which symptoms is it to have effect? These questions are as old as knowledge of the immune system itself and just as relevant today as they were. It is true that our information on the cellular and humoral components of the immune system and our insight into the relevant regulatory systems have increased impressively, yet our wish for a pharmacological means of influencing the immune system has only in part been fulfilled. The various theoretical possibilities of treating tumor diseases by influencing the immune system actively or passively, specifically or unspecifically by means of different groups of substances are listed in Table 1. The possibilities are based on the assumption that tumor illnesses are caused or favoured by incorrect or insufficient control by the immune system. Illnesses such as autoimmune diseases, hypersensitive reactions, or immune complex illnesses due directly to a wrongly directed immune response are still considered as possible additional indications, as well as infectious diseases influenced in their occurrence, severity and course by.an insufficient body resistance. To date it has only been possible to provide conclusive evidence for a clinical effect of certain immunomodulators (vaccines and antibodies) in infectious diseases, and here mainly in prophylaxis [1, 2]. In recent years organ transplantation has arisen as an additional indication for immunosuppressive immunomodulators. In particular in this field successes were achieved by the use of substances inhibiting cell proliferation such as antimetabolites (purin antagonists such as azathioprine) alkylating agents (cyclophosphamide amongst others [3, 4]), hormones (corticosteroids [5, 6]), and with drugs which selectively affect only the immune system, such as antilymphocyte globulin [7, 8] (e.g. Pressimmun<~), monoclonal antibodies against T-lymphocytes [9], cyclic peptides (cyclosporin A [10]) or guanidinic derivatives [l l] like _+15-Deoxyspergualin. In the case of immunosuppressive drugs questions remain as the actual mechanisms involoved; however, clinical studies have already shown a clear therapeutic effect. With respect to drugs suppressing the immune system specifically due to the clinical effect, we are thus in possession of substances which can be used as standards in models to predict clinical effectiveness, in order thus to ascertain other substances of advantage with regard to effect and tolerability. The situation is a different one for illnesses such as Table I. Possibilities for tumor immunotherapy Unspecific

Passive Mediators Interferons

Specific

lnterleukins Lymphotoxins Tumor necrosis factors Colony stimulating factors (CSF) Complement factors K-cells; NK-cells Antibodies against tumors --lgG2~; IgG~ (mouse) --radioactive labeled antibodies --immune cytostatics; immunotoxins Lymphocytes --specific sensibilised T-cells

Active Immunostimulants Microorganism and microbial products Synthetic chemoimmunotherapeutics

Tumor cells + adjuvant tumor cells + neuraminidase tumor cells + BCG tumor antigens + adjuvant Hybrids of tumor cells and normal cells Virus infected tumor cells

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autoimmune diseases, chronic infectious diseases and tu.mor illnesses. Here, too, immunomodulators have already been employed, characterized by the fact that their effect was either passive or active with regard to the receiver, or specific or unspecific depending on the relevant antigen. However, thus far it has only been possible to provide evidence of a clinical effect for some of these substances. It is not the aim of the present publication to discuss the possibilities and problems of a passive influencing of the immune system, that is to say substitution in an organism with humoral or cellular components of the immune system, such as with antibody preparations or mediators (e.g. y-interferon; interleukin I and II; colony stimulating factors) or lymphocytes. Neither is it our intention to deal with the effectiveness and problems of active specific immunostimulation, as is practised using vaccines, for the prophylaxis of infectious diseases, and adjuvants [1], even if there are both experimental and clinical indications of a therapeutical effect of specific immunological tumor therapy in certain tumor illnesses and under certain conditions [12]. The present publication will limit itself to the discussion of chemoimmunotherapeutic drugs with particular regard to active (antigen-) unspecific immunostimulation. Results achieved so far with this kind of immunomodulation are summarized and discussed and future prospects presented.

Historical development Active unspecific immunostimulation is regarded as a pharmacological influencing of the endogenous functional capacity of the immune system, not supplemented by substitution. Its aim should be to alter the either insufficient, or even normal reactive state of the immune system in such a way that, independent of the antigen being tested, a normalisation or increase of the primary or secondary immune reaction takes place. This independence of the respective antigen thus constitutes the essential difference from active (antigen) specific immunostimulation. In the latter an increased immune reaction against only one particular known antigen is desired. This independence of the antigen also determines the area of indications for active unspecific immunostimulation: basically this embraces all illnesses immunologically mediated or which can possibly be immunologically influenced, and in which the antigen connected with the illness varies from person to person or is not or insufficiently definable. The first attempts to actively stimulate the immune system unspecifically go back over 100 years. In 1851 Latour [13] tried to influence tumor growth with living bacteria (Treponema pallidum) and following this Busch [14] attempted the same with erysepela. At the beginning of this century Adam [15] and Much [16] developed bacterial preparations to influence infectious diseases immunotherapeutically. Concentrates, toxins, lysates of bacteria, proteins, lipids and endotoxins were employed for treating tumor and infectious diseases (see Table 2). Research in this field reached its peak in the twenties and thirties of this century, but was however subsequently overshadowed by the development and successes of chemotherapy and antibiotic therapy. In the past fifteen years there has been a revival of work in the field of immunostimulants, partly due to the increasing occurrence of antibiotic and chemotherapy resistant bacteria, but largely in the hope of acquiring an additional instrument in the thus far only partially successful fight against tumors with the aid of immunostimulants. This revival has been accompanied and

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H. H. SEDLACEK et al. Table 2. History of immunomodulators 1851 1890 1893 1914 1919 1921 1924 1956 1969 1972

Latour Buchner Coley Adam Much Calmette+ Guerin Buzello Johnson et al. Math6 Halpern

Living bacteria (Treponema pallidum) for tumor diseases [13] Compressed fluids of bacteria produce leucocytosis [17] Toxins (Serratia marcescencs toxins) for tumor therapy [18] Bacterial preparations (infectious diseases) [15] Mixture out of bacteria lysates (infectious diseases) [16] Culturing of Bacterium-Calmette-Guerin (BCG) [19] Proteins [20] Endotoxins (adjuvants) [211 Unspecific tumor immunotherapy with BCG [22, 23] Unspecific tumor immunotherapy with C. parvum [24]

supported by the extremely large increase in our knowledge of the immune system network in the past three years. Thus Old et al. [25], Math6 [22, 23, 26] and Halpern [24] all recommended the administration of bacterial whole germ preparations as immunostimulants for the therapy of tumor illnesses. Other substances followed, sometimes purified components from bacteria or fungi, sometimes antibiotics or chemically synthetic preparations (for a list of such substances see Table 3) [27]. These, too, were developed mainly with the aim of influencing tumor growth immunologically. They were employed and tested for effectiveness in infectious diseases, if at all, only complementarily and usually only in prophylaxis. The correlation of preclinical and clinical results

Both the whole germ preparations (Corynebacterium parvum, picibanil, BCG and others) and those isolated from bacteria or fungi or those substances produced synthetically were selected because of their immunostimulatory activity. The number and/or function of Tand B-lymphocytes, macrophages, natural killer cells and/or granulocytes in various in vitro, ex vivo or in vivo test systems were parameters for immunostimulation [summaries in WHO Report 28, 29-37]. For this reason studies were carried out in allogenous and Table 3. Immunostimulators Bacterial preparations Corynebacterium parvum Picibanil (Streptococcus pyogenes)

BCG (Bacillus Calmette-Gu6rin) Bordetella pertussis

Defined components or products of bacteria MER (BCG) Cord factor and synthetic analogs (BCG) MDP and synthetic derivatives (BCG) Biostim (glycoprotein of Klebsiella pneumoniae) Cell wall proteins (Nocardia, BCG) Lipid A

Defined components of fungi Lentinan (Polysaccharide of Lentinus edodes) Krestin-PSK (protein-bound polysaccharid of Cariolus versicolor) Schizophyllan (branched Glucan of Schizophyllum commune) Glucan (long chain fl 1-3 Polymannan of yeast) Mannozym (Polymannan + Polyglucan of yeast)

Antibiotics Bestatin Forphenicinon Therafectin Amphotericin B Synthetic compounds Levamisol Diethyldithiocarbamat (DTC) Amygdalin (Lactrile) Phenyl-trichloroethoxycarboxamitdothiazol (F 1686) lsoprinosine NPT 15392 Aminoethyl-phenyl-piperidinhydrochlorid (CP 46665) N -carboxyphenyl-chloranthranillin-acid (CCA) Dimethylhydrazin Azimexon (2-Cyanaziridine-analogs) Lysolecithin and analogs Polymet hylmethacrylate Poly-l-C Pyrancopolymer Polynucleotide Duplexe Tuftsin

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syngenous tumor models, mostly of the mouse, rat and guinea pig. These were exclusively transplantation models of spontaneous origin, viral or carcinogen induced, partially metastasizing tumors. After treatment of the receiver animals with immunostimulant whole germ preparations or with chemically defined substances a clear increase in NH2

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resistance against subsequently transplanted tumors could be shown in these tumor models, that is to say the growth of the subsequently transplanted tumors was impaired. Additionally to this prophylactic effect a therapeutic effect could be found; after treatment of the tumor bearing animal tumor growth was seen to be impaired. These experimental data led to the hope that a breakthrough could be made in clinical tumor treatment with the aid of active unspecific immunostimulation (summary in Seshadri and Podural, [38]). After the first promising clinical data [26, 39] with whole germ preparations the clinical studies subsequently performed produced only disappointing results [40-45]. As a consequence attempts were made to isolate, identify and synthetise the immunopharmacologically active components of whole germs with the aim of increasing the immunostimulant activity of the preparation and reducing its toxicity. Although these purified or synthetic structures were also very active in preclinical test systems, most of the substances have so far failed to show any clear effect on tumor illnesses in clinical studies. The picture was a similar one for the substances then isolated from fungi, for antibiotics and various synthetic substances. However, a series of substances is still

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Table 4. Possible reasons for clinical ineffectivity of chemoimmunotherapeutics in clinical tumor diseases Screening system too sensitive (selects too many false compounds) Test models (immunopharmacological models, tumor models) inadequate with respect to clinical tumor immunotherapy N o relationship between the immune response and tumor disease Clinical tumor disease cannot suli~cicntlybe staged to evaluate minor therapeutic effect of chcmoimmunotherapcutics Effect on iatrogcn induced, immune mediated diseases in cancer patients has been neglected Immunopharmacokinctic data (dose-response relationship; application schedule; therapeutic index) has been neglected

undergoing clinical trials, so that it is as yet not possible to make a final judgement on them. Nevertheless, an evaluation of the numerous already completed prospective and randomised clinical studies must lead to the conclusion that, to date, not one active unspecific immunostimulator has been found to be systemically and reproducibly thera-. peutically effective in tumor illnesses. When administered locally, that is into or in the vicinity of a tumor, regression has been found in some types of tumor (for instance skin melanoma) or there has been a prevention of local relapses (for instance in carcinoma of the bladder), there has, however, been no significant effect on the systemic tumor illness [46-49]. There was no correlation at all between the, all in all, negative clinical results and the positive preclinical data in different tumor models. Thus it must be concluded that the preclinical test systems used to ascertain active unspecific immunostimulants for tumor therapy have no clinical predictive value. Several reasons may be given for this contradictory situation (see Table 4). But these reasons do little else than illustrate our present situation; we know too little about the kind of interaction between tumor cells and the immune system and the significance of this interaction. We are familiar with some sections of this interaction from studies on experimental tumors, but the relation of these sections to the whole complex system, or whether these are simply coincidentally selected phases of continually changing reactions and differing balances of ever-changing reaction partners is not known. Similarly, we do not know whether the few parameters familiar to us from the tumor-host relationship in experimental tumor systems are also present in autochthonous spontaneous tumors, that is in patients, or whether they correspond in significance to those of transplantation tumors. In the case of spontaneous autochthonous tumors other parameters, possibly unknown or inaccessible to us, may play a crucial role in the interaction between tumor and immune system. Thus we also do not know whether these are at all present in our experimental tumor systems or can be recorded by them. For these reasons alone the search for active unspecific immunostimulants for tumor therapy may have been unsuccessful thus far, and they may explain the contradictions between the results in preclinical models and clinical studies. Moreover our albeit limited, knowledge of the interaction between tumor and immune system already offers some possible explanations for the failure of active specific immunostimulation in tumor therapy (see Table 5). On the other hand, on the basis of this knowledge, it is to be feared that with this kind of tumor therapy immunological reactions may provoke an increased tumor growth [50, 51]. All these interpretations of the failures in clinical testing of active unspecific immunostimulants are based on the assumption that immune surveillance of a tumor cell [52] is a demonstrable hypothesis. However, it must not be forgotten that this hypothesis may not apply or only applies in a limited number or tumor types (e.g. in virus-induced tumors), and that thus the immune system is not causally involved in tumor development or growth in a number of tumors not belonging to this group. Consequently the immune reactions

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H. H. SEDLACEK et al. Table 5. Escape of tumor cells from immunological destruction Host

Genetic background (appropriate Ir genes) Tolerance (perinatal?) Immunosuppression (carcinogens, co-factors) Immunodeficiency (age, disease) Lack of immunologically competent cells Immune complexes Suppressor cells Stimulating antibodies Tumour

Weak immunogenicity Change of antigens (masking, modulation, shedding) Selection of resistant clones Secretion of immunosuppressive substances

and parameters to which tumor control functions have been attributed would be nothing other than epiphenomena. In the face of these tumor therapy failures, ought the project "active unspecific immunostimulation" be abandoned? In order to answer this question the experience of the NCI--U.S.A. in the development of chemotherapeutic drugs for tumor therapy should be considered. Over 400,000 different substances were tested in transplantation tumors, and of these 600 have been found to be therapeutically effective in various experimental tumors (leukemias and solid tumors). Of these 600 substances less than 30 have also been found to be clinically effective, mainly in leukemia and only to a small extent in solid tumors [53, 54]. In the majority of human tumor illnesses chemotherapeutic drugs do not have any significant life-prolong effect, even though the drugs used were chosen for clinical studies because of just this effect in corresponding experimental transplantation tumors. Thus in chemotherapy, as in active unspecific immunostimulation, there is the problem of the predictive value of preclinical tumor models. Despite all reservations, however, the successes of tumor chemotherapy are evident. The price which had to be paid for this has been a costly suitability testing for an unprecedented number (>400,000) of substances. Since to date a considerably smaller number of substances has been tested for active unspecific immunostimulation, there is still a chance, despite the discrepancy between the informational values of the preclinical and clinical models, of finding a clinically effective preparation during the testing of a larger number of substances. On the other hand, a revision of the preclinical models on the basis of our experience to date, with the necessary corrections, ought to increase the chances of finding an immunostimulant. Similar processes are also underway in the development of chemotherapeutic drugs [55].

Improvement of the screening system Since clinical testing of active unspecific immunostimulants has not yet produced any positive results, we are not in possession of a standard substance with which we would be able to test the informational value and the quality of a preclinical system of selection. Any compilation or alteration of such a test system can only be done on the basis of insights into the organisation of immunological reactions, taking into consideration the preceding negative results in the development of active unspecific immunostimulants. In accordance with our knowledge of the immune system a substance would have to be able to influence (i.e. activate) immune system cells in number and function, such as

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macrophages, granulocytes, lymphocytes, natural killer cells and subtypes of these cells in order to be considered as an immunomodulator. On the other hand, this activation ought to be pharmacologically evaluable in an immunologically mediated experimental illness. The necessary illness models for testing the suitability of an immunomodulator have to have predictive value for the clinical situation. This, however, is tantamount to working in a black box which is only illuminated by the mistakes we have recognized and are able to avoid in the future. One of these mistakes was, as we now know, that we carried out studies on transplantation tumors which only have limited, if any, informational value for the clinical situation. It remains to be seen whether time consuming testing with experimentally induced autochthonous tumors (such as those induced by u.v. radiation or carcinogens), as has been begun by NCI [56], will provide more useful information. It is well known that such tumors develop after a relatively short latent period and are more strongly immunogenic than spontaneously arising autochthonous tumors in animals or humans. Thus here, too, there is a danger of false-positive results. A further mistake was that in the preclinical models the influence of active unspecific immunostimulants was recorded mainly in primary tumor transplants and to a lesser extent in spontaneous metastasation (especially in the stage of minimal residual disease) and/or that the survival chances according to the degree of metastasation in experimental tumors in a manner of relevance for the clinical situation were neglected. Such experimental data, correlated to immunokinetic studies on the dose-effect relationship, its time dependency and on the development of refractory phases after repeated administration to the respective cell of the immune system activated would have provided a useful basis for finding a safe immunostimulating dose in humans and evaluating its therapeutical value. But it is the immunokinetic determination of the effective dose and of an effective application scheme which has been largely neglected in the past. A further grave mistake in the development of active unspecific immunostimulants so far has been the general restriction to tumor illnesses and the consequent smaller regard for other indications during testing. This one-sidedness was implemented, even though the connections between tumor growth and immune system are not clearly understood and no one at present can estimate to what extent and via which mechanisms the immune system is capable of influencing the growth of spontaneously developing, autochthonous tumors. The inclusion of additional models into the screening programme, in which the immune system is more obviously involved in the occurrence of the illness than is the case in tumor illnesses, could enable us to make a wider and more appropriate assessment of the therapeutic value of active unspecific immunomodulators. In this context chronic infection models, chronic inflammation models and models for autoimmune illnesses ought to be discussed. Substances which show effect in these models would be likely candidates for clinical testing, and positive results in the clinical situation would help to assess the predictive value of these experimental models. In view of our inadequate knowledge as to which cell in the immune system is to be activated by an active unspecific immunostimulant in order to be of therapeutic value for certain symptoms, we find it advisable to plot the immunomodulatory activity of a test substance as widely as possible, that is to say to assess its effect on the function of B-cells, T-cells, NK-cells, granulocytes and macrophages. Any arbitrary limitation of the screening system to a selected number of test models reduces the chances of finding a substance which is effective both in the preclinical model and in the clinical situation. For testing, e x vivo

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H . H . SEDLACEKet al.

and in vivo animal models treated with the test substance should be preferred to pure in vitro models in order to avoid in vitro artefacts. Considering this previous experience, we devised a screening system in two phases [36, 57-59] (see Fig. 1). In the first phase the activity of the test substance on T-cells, B-cells, NK-cells, macrophages and granulocytes is ascertained in various function tests (see Fig. 1). Ex vivo and in vivo tests are carried out in mice and in vitro tests for human cells (monocytes and granulocytes). A precondition for the parenteral administration of the test substance is that it is free of endotoxina. Endotoxin contaminated substances are excluded from the tests or administered perorally, or parenterally in CH3/HcJ-mice genetically resistant against LPS. Substances which are able to influence the function of one or more cells of the immune system (i.e. increase or reduce them) are subsequently tested in a second phase in a so-called suitability model. These include infection models, inflammation models, models for autoimmune illnesses and organ transplantations, as well as tumor models. It is hoped that these models will have predictive value for the clinical situation. A special importance is attributed to infection models [59]. In these infection models the increase in resistance due to the immunostimulator against a subsequent i.v. injection of an infectious and lethal dose of various bacteria or viruses is tested (prophylaxis tests). Parallel tests are carried out on immunosuppressed animals to record, in the model, the iatrogenously induced immunosuppression in the clinical situation (e.g. due to cytostatic drug treatment). Further tests show whether the respective immunostimulant is able to influence an already present chronic, bacterial and fungal infection therapeutically. Such infection models have recently been set up in our laboratories [59]. At present we have chronic infection models induced by Salmonella typhimurium, E. coli, S. aureus, Listeria monocytogenes, Bacteroides and Candida albicans. Apart from these infection models studies are carried out to find out whether these test substances have a therapeutic effect or can enhance the course of the illness in humorally (hemolytic anemia or immune complex nephritis in mice) or cellularly (experimental autoallergic encephalomyelitis in rats) mediated experimental autoimmune illness or a locally induced inflammatory reaction .[57]. An enhancement of the illness could be an indication of intolerability for the test substance and thus for a greater risk of side effects in the clinical situation. Tests in organ transplantation models (skin transplants in rats and allogeneous tumor transplants in mice).show whether the substance could be of value as an immunosuppressive drug in organ transplantation. Despite their questionable, or not yet proven, predictive value we have retained immunostimulant tests on experimental tumor models: However, we have concentrated our efforts on the question whether a test substance is capable alone, or together with cytostatic drugs, of influencing the growth of spontaneously arising micrometastases after surgical removal of the respective transplanted primary tumor. We base our investigations on the assumption that the immune system only has a chance in immune surveillance of tumors, if at all, when the tumor mass to be resisted by it is as small as possible. A clear and reproducible thei'apeutic effect demonstrable in several syngenous tumor models, i.e. a test substance mediated prolongation of the survival time, would appear to us to be a sound basis for clinical testing, despite all reservations. With similar reservations, and as a reminder that careful use should be made of these substances in clinical tests, we see the metastasis growth accelerating properties of substances. All in all, it is our aim to perform as wide experimental tests on immunostimulants as possible. This is in some contrast to the work of the National Cancer Institute in the

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I-I. H. SEDLACEK et al.

Table 6. Requests for chemoimmunotherapeutics Defined, stabile, standardizable compound Effective in relevant models, way of action known No paralysis of immune system, risk of overstimulation should be small No antigenicity, no cross reactivity with human tissues No side reaction by activation or acceleration of enzyme cascades (complement, blood clotting system) No or a negligible toxicity Compound should be metabolised by the body No carcinogenicity

U.S.A., where immunomodulators are preferred, or testing is only done with regard to an antitumoral effect [60]. Both study groups, however, attach the same importance to toxicity testing of active unspecific immunostimulants; and this with an eye to at least reducing the risk of intolerance reactions to a minimum for the uncertain and still questionable clinical-therapeutic effects of immunostimulants. The WHO demands [28] for such drugs can be taken as guidelines (see Table 6). RESULTS The screening system described above has been used by us for about 5 years, and is continually being enlarged with essential function tests and suitability models which appear to us to be relevant, especially the chronic infection models. Over this period a number of reference substances have been tested, e.g. the familiar bacterial whole germ preparations, substances purified of them or originating from them, such as muramyldipeptides, preparations from fungi such as various glucans and several synthetic preparations. With only a few exceptions, all these test substances were able to activate macrophages in vitro. A part of those compounds was also active in vivo and was able, in part, to enhance various lymphocyte functions, or, in the case of immunosuppressors, to inhibit them. Although there were quantitative differences, it was difficult to distinguish the qualitative effect of each substance on the immune system. On the other hand, we had the results from the suitability models, especially the chronic infection models. Here a large majority of the substances demonstrated no therapeutic effect. One portion led, with reproducible results, to an aggravation of the chronic bacterial infectious diseases and to a shortening of the survival time. Another portion of drugs was therapeutically effective in the one and enhancing in another bacterial disease; in only a few cases it was possible to find substances with a therapeutic influence on the course of the chronic infection (Table 7). Depending on the substance, different therapeutic effects could be seen with different types of germ. A substance such as bestatin, effective in chronic E. coli or Salmonella Table 7. Chemoimmunotherapeutics--Screening for immunostimulation (1980-1985) % % Number of Related to all Related to compounds compound immunoactive compounds Tested altogether Immunopharmacology (ex vivo) Active Therapy of chronic infections

Inactive Enhancing Active/enhancing Active

> 500 26

5.2

14 4 2 6

2.8 0.8 0.4 1.2

53.8 15.3 7.7 23.0

Chemotherapeutics

111

typhimurium infection, was ineffective in such infections as Staph. aureus or Candida albicans. Staph. aureus infection could be influenced therapeutically by another immuno-

stimulant, schizophyllan [59, 60]. In contrast, both bestatin and schizophyllan quantitatively and qualitatively activated immune system cells in similar ways. In their activity (that is to say their activity on immune system cells recordable with our test models) they were qualitatively not distinguishable from substances which enhanced chronic infections or which showed no effect in these models (Table 8). On the basis of this information it must be concluded that the activation of immune cells recorded with this screening system says little about the pharmacological, therapeutical value in infection models, for instance. Since the screening system employed includes all the immunological test procedures known to date, we must further conclude that we as yet are not in possession of any immunological parameter of predictive value for immunotherapeutic effect in these (immunologically mediated) chronic infection models. On the other hand, these models enable us to distinguish between therapeutically effective and ineffective (illness enhancing) substances. Although unproven, it is likely that the different effects in the infection models are caused by a substance mediated, differing influence on the immune system, which can be either supportive (immunotherapeutic) or damaging (immunotoxic) or immunosuppressive. Thus with chronic infection models immunotherapeutic effects can be differentiated from those which altogether are immunotoxic or immunosuppressive. In contrast, these effects have not been able to be distinguished as yet either by simply testing for activation of cells of the immune system or by additional protective (prophylactic) tests. One of the few substances with therapeutic effect in our chronic infection models is bestatin (2S, 3R) 3-amino-2-hydroxy-4-phenylbutanoyl-e-leucin, an inhibitor of leucinaminopeptidase and aminopeptidase B, isolated from Streptomyces olivoreticuli by Umezawa et al. [62]. Below the informative value and the problems involved in effectivity and suitability testing in active unspecific immunostimulants will be discussed taking the immunopharmacological results obtained with bestatin as an example. Bestatin is free of endotoxins and was added to cells in vitro at a dose range of 50-400 ~g/ml (the optimal effective dose was 100 #g/ml) or administered i.p. or i.v. or p.o. to mice at a dose range of 0.5-50 mg/kg (optimal dose 5 mg/kg). This dose is far below the cytotoxic or acutely toxic dose of LDs0 > 600 mg/kg for mice. Studies on the influencing of the immune system (see Tables 9, 10) have shown that bestatin clearly activates macrophages in vitro (mouse and human) as well as in vivo (mouse) [57]. This activation was accomplished by an increased formation of oxygen radicals, enhanced pinocytosis, cytotoxicity against tumor cells, the formation and secretion of lysosomal enzymes as well as the formation and secretion of prostaglandins (PGE 2 and PGF2). If at all, granulocytes and B-lymphocytes are only negligibly affected. The affect on T-cells differs. The activity of cytotoxic T-cells (CTL) either remains unchanged or is diminished by bestatin. The delayed type reaction (DTH) against various antigens is, however, clearly enhanced, as well as a local graft versus host reaction, measured in a popliteal lymphnode assay (PLN). The immunopharmacological range of bestatin did thus not principally differ from that of a number of other substances, such as BCG, C. parvum or glucans. During testing in suitability models (see Tables 11, 12) bestatin was found to be clearly effective, especially in infection models, both in prophylaxis and therapy experiments [59].

+ + + +

Polysaccharides Heteroglycan LPS + LPS branched Glucan Glucan

+ : reduction of infectivity. - : enhancement of infectivity. O: no effect.

(killed material)

Bordetella pertussis

+

+

Bacterial prep.

M0

Bestatin

+

+ 0 (+ ) 0

+

T-cells (DTH)

(+) (+) +

0

0

+

S. typh.

+

0

0

St. aur.

0

Li. too.

+

E. coli

Chronic infection (therapy)

0

Anaer.

0

0

Cand.

Table 8. Efficacy of immunostimulating drugs on infection models

B-cells (antibody)

Immunopharmacology

Peptides

Compound

+

0

0

+

E. coli

St. aur.

0

Ps. aer.

Sepsis (prophylaxis)

+

0

KI. pneu.

e~

>

113

Chemotherapeutics Table 9. Effectivity of bestatin

Doses tested:

T-cells

in vitro in vivo

CTL DTH

B-cells

PLN AB PFC

50-400 # g/ml 0.5-50mg/kg (i.p.;i.v.; p.o.)

Optimal ED Optimal ED

100#g/ml 5 mg/kg

(C57BI/6-DBA2/J) (DBA 2/J-ESb-Lymph.) (NMRI-SRBC) (NMRI-S. typhimurium Ag) (intermittent injections > daily injections) in vivo (Lewis into Lewis-Brown-Norway F~) ex vivo (NMRI-SRBC) (NMRI-S. typhimurium Ag) ex vivo (NMRI-SRBC) in vitro ex vivo in vivo

0/0/+ + + 0 0 0/+

+ : stimulation. - : suppression. O: no effect.

In these infection models it was astonishing that the effect was limited to some bacterial germs and that during prophylaxis experiments a protective effect was to be found with different types of germs than the therapeutic effect in chronic infections. With the limited evidence provided by these models we must conclude that results obtained on the prophylactic effect of an immunostimulant on a certain germ do not allow us to project the therapeutic effect of the same immunostimulant onto a chronic infection caused by this germ. Bestatin did not cause an enhancement of the illness in any of the different infection models. Thus there were no signs of an immunotoxic effect. Repeated applications of bestatin were particularly effective when they were administered at intervals of 2-3 days. Daily applications produced smaller effects [59]. The same could be observed for the activation of macrophages and stimulation of D T H [57, 63]. Here, too, administration at Table 10. Effectivity of bestatin in vitro in vivo

Doses tested:

50-400 g g/ml 0.5-50 mg/kg (i.p.; i.v.; p.o.)

Optimal ED Optimal ED

100 #g/ml 5 mg/kg

Immunomodulation I1 NK-cells

NK

e x vivo

Macrophages

Of

in vitro e x vivo

Pinocytosis

in vitro

CTM

ex vivo in vitro e x vivo

LYSOS.ENZ. (fl-Gluc.; fl-Galact. N-ac.-Gluc.) PGE2/ PGF2~ Granulocytes

Oi NBT

+ : stimulation. - : suppression. O: no effect.

in vitro e x vivo

(CBA/JCR-YAC-1) (DBA 2/7-YAC-1) (NMRI; human Mo) (NMRI) (NMRI, human Mo) (NMRI; 198Au) (NMRI; Nu/Nu; human Mo) (NMRI; Nu/Nu; C3H/HeJ) (-K562; YACI; 721; LS; ESb) (NMRI; human MO) (NMRI) (Synthesis + Exocytosis)

(NMRI; human Mo) (NMR1) (Synthesis + Secretion) (intermittent injections > daily injections) in vitro (human PMN) ex vivo (NMRI) in vitro e x vivo

(+) 0 + + + + + + + + + + 0

(+)

114

H . H . SEDLACEK el al. Table 11. Effectivity of bestatin

Doses tested:

0.5-50 m g / k g (i.p.; i.v.; p.o.)

in vivo

Optimal ED 5 m g / k g

Models of aptitude 1 Tumor

--primaries: (growth)

Sarcoma 180 ESb; Lewis L.; BI6; R3230 ESb-Lymphoma

--metastases: (number)

Infections

i.p./i.p, s.c./i.p, s.c./i.v.

Lewis L.; BI6; R3230 Candida alb. Staph. aureus various g r a m ( + ) g r a m bacteria Herpes simplex ( + EAG)

--acute: (mortality)

----chronic: (germ number, inflammations)

E. eholi 0 4 : H 5

S. typhimurium

day 1-10 day 1-10 10 × before resection day 1-10

s.c./i.v, s.c./i.v.

10 x after resection 5 × ip (-10-0) -1/0/1 -1/0/1

i.v./p.o. i.v./p.o. i.v./p.o. i.c./i.p. i.c./i.p.

1 × (d-16) 9 x (d-16-0) every 2nd day 7 × (-6-0) 7 x (0-6) 7 x (7-14) 3 x (-17-15) 4 x (-1-2) 7 x (0-12) X x (0-~) X x (4-~) 2 x (0, 4) 3x (0,4,8)

i.v./i.p. or i.,v./i.v. i.v./i.p. or i.v./i.v.

Candida alb.

i.v./i.v.

Staph. aureus

i.v./i.v.

Bacteroides OCragilis + melaninogenieus ) Listeria monocytogenes

i.v./i.p. i.p./i.v. or i.p./s.c. i.v./i.v,

-0 0 0 --0 --

-----

0 0 0 0

x x (o-,.b)

o

9 x (3 19) every 2nd day

0

11 x (0-20) every 2nd day

0

- - : reduction of t u m o r growth or infectivity. 0: no effect. D,Iq: administration of bestatin until death of the animals.

Table 12. Effectivity of bestatin Doses tested:

0.5-50 mg/kg (i.p.;i.v.; p.o.)

in vivo

Optimal ED 5 mg/kg

Models of Aptitude II Autoimmune diseases

Anemia (antibody; RBC; lifespan)

G v H (C57B1/6 grafted to C 3 H x C 5 7 B 1 / 6 Fj)

i.p,

IC-Glomerulonephritis (proteinuria; lifespan)

G v H (DBA/2 grafted to C57B1/6 x D B A 2 F~)

i.v.

Spinal cord preparation (G.P.) + B . P . into Lewis rats (Lewis skin on Fisher rats)

i.p.

i.p.

+ALG

i.p.

Zymosan; collagen

i.m.

Encephalomyelitis (onset of dis.; lifespan) O r g a n transplantation Skin (Survival)

Inflammation

M u s c u l a r tissue knee joint (swelling; histology; enzymes)

almpairment of the A L G induced prolongation of graft survival time.

i.v.

1 0 x (0-9) 1 0 x onset of disease 14x (0-t3) 14× onset of disease 10 × (0--9)

0 0

29x 10x 10× 10x 10 x 3×

0 0 0 ----

3 ×

(10-38) (-9~) (~9) (-9-0)" (0-9) a (0-2) (0-2)

0 0 0

--

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115

intervals was more effective than daily administration. It would thus seem logical to see a direct causal relationship between the therapeutic effect of bestatin in infection models and macrophage activation and stimulation of DTH. Bestatin demonstrated antitumoral effects only in a few tumor models of our studies and only under certain conditions: the growth of the primary tumor transplant was not impaired, or only after local intratumoral application in the i.p. growing tumor (nonmetastasizing sarcoma 180). Influence on metastasis growth could be demonstrated in BI6 melanoma and in ESb lymphoma. Since, in ESb lymphoma, metastatis is accompanied by the appearance of cytotoxic T-lymphocytes, the possibility cannot be ruled out that there is a direct relationship between the bestatin induced decrease in CTL and metastasis reduction. Despite its immunostimulatory activity bestatin did not have any effect on humorally or cellularly mediated experimental autoimmune illnesses, but it did clearly reduce chronically productive inflammatory reactions after intramuscular or intraarticular injection of complement activating substances such as zymosan and collagen. As a direct influence of bestatin on the complement system can be ruled out, it remains for use to speculate as to the relationship between this anti-inflammatory activity of bestatin and its ability to activate macrophages. It may be that the bestatin-induced increased secretion of prostaglandins from macrophages is of significance in this antiinflammatory activity. It is, however, unclear why not adverse but rather inflammation promoting effects dominate, such as the release of lysosomal enzymes from macrophages. Our studies on skin graft models have clearly underlined the immunostimulatory activity of bestatin: Bestatin is able to neutralise, in part, the immunosuppressive effect of antilymphocyte globulin (measured by the prolongation of transplant survival time). The preclinical data from these bestatin tests were the basis of the clinical tests at present underway, mainly on selected chronic infections but also on chronic inflammatory illnesses and on minimal residual tumor diseases. The results of these clinical studies will show whether the preclinical models, especially the chronic infection models, can be regarded as being of predictive value for the clinical situation. Should it be possible to show a reproducible clinical success, we would be in possession of a clinically therapeutically effective, active unspecific immunostimulant, which could be used as a standard in the search for further preparations of this class of substances. FUTURE PROSPECTS The development of active unspecific immunotherapy so far has been mainly impaired, as our experience has shown, by our insufficient knowledge of the relevant, possibly varying, regulatory systems of immune reactions in both experimental and clinical illnesses (for conclusion see Table 13). Whether the defects in preclinical models correspond to those in clinical illnesses is not known. We do not even know the extent of immune system insufficiency in the different cases or how it is involved in the course of illness. With symptoms which, seen from the point of view of immunology, are, if at all, insufficiently characterized, we are testing substances whose immunopharmacological activity we are not able to describe sufficiently due to our lack of methods and knowledge. Thus we should not wonder at our failures of the past. Yet it would be a mistake, as a result of these setbacks, to say there is no future in the project on active unspecific immunostimulation. But we would also be making a mistake if we kept to the preclinical selection procedures of

116

n . H. SEDLACEK et al. Table 13. Chemoimmunotherapeutics

Conclusions A considerable number of compounds are able to activate cells of the immune system Up to now, in Western countries, not one compound has successfully passed clinical examinations Thus activation of any cell of the immune system in experimental models does not say anything about the therapeutic effect in patients Needed are predictive models to differentiate between immunotoxic and immunotberapeutic effects (chronic infection models and others) Estimation of any compound can only be done after a battery of immunological and therapeutic tests have been performed This needs a lot of specific scientific and economic investments Facing the up to now worldwide negative outcome of research on chemoimmunotherapeutics it has to be defined as a high risk project

the past. Our slowly acquired experience in this field can at least, when adequately considered in the test models, be of further help in the selection of substances. For us this means: widescope testing of a test substance as to its functional affect on the immune system cells, and subsequently testing the effective substances in suitability tests with a probable clinical relevance and an assumed or proven immunological context. As far as we know, these conditions do not apply for experimental tumor models. Thus other, more suitable models will have to be developed in other indication areas. These models, to which chronic infectious illnesses can be counted, would be more likely to answer the question of a useful immunopharmacological effect and the problem of immunotoxic side effects than functional tests on cells of the immune system. Here we are only able to test whether a substance stimulates or suppresses cell functions. In the case of activation these cell function tests are not (yet) able to tell us whether the activation is immunopharmacologically useful or immunotoxic. Such a time consuming screening system can clearly only be possible for a limited number of substances. Past experience in other fields, for instance the development of cytostatic drugs, has shown us that it is of the utmost importance to test a large number of different structures in order to have a chance of finding an effective substance and subsequently, by means of structure variations, of optimizing the relationship of effect and side-effect. The immunostimulating substances tested so far were not able to fulfil the conditions set up in Western countries for clinical effectiveness. Assuming that they have been adequately tested, it would be of no use to vary their structure in order to find more effective analogues. In future we shall have to test new structures for immunomodulation "blindly", either with traditional and time consuming immunological function tests or with selected test systems reflecting certain sections of the regulatory systems of immune reactions, such as the expression of membrane antigens, of receptors for certain mediators and receptors. The questionable significance of these sectional pictures for the overall function of the complexly regulated immune system would be compensated by the advantage of being able to test a large number of substances relatively simply and quickly. Selected effective preparations could then be broadly tested in various test systems for stimulation of immune system cells and suitability. With increasing knowledge of immunomediators and their primary sequence this procedure would have the additional advantage that we would then be able to examine analogous preparations of mediators specifically for immunopharmacological effectivity. A further aspect would be to select substances with particular regard to their influence on cell membrane metabolism, and thus to find substances able to modulate the immune

Chemotherapeutics

117

system. This was first done by Umezawa and co-workers [62] in the course of their search for inhibitors of cell membrane associated enzymes. In the course of this work bestatin was found to be an inhibitor of leucin-aminopeptidase and an immunostimulant. The future will show whether this preparation will stand clinical trials. REFERENCES 1. Edelmann R. Vaccine adjuvants. Rev. infect. Dis. 2, 370 (1980). 2. Schwick H. G. and Haupt H. Chemic und Funktion der Humanplasmaproteine. Angew. Chem. 92, 83-95 (1980). 3. Winkelstein A. Mechanisms of immunosuppression: Effects of cyclophosphamide on cellular immunity. Blood 41, 273 284 (1973). 4. Calne R. Y. Pharmacological immunosuppression in clinical organ grafting. Observations on four agents: Cyclosporin A, Asta 5122 (cytimun), lambda carrageenan and promethazine hydrochloride. Clin. exp. Immun. 35, 1-9 (1979). 5. Butler W. T. and Rossen R. D. Effects of corticosteroids on immunity in man. I. Decreased serum IgG concentration caused by 3 or 5 days of high doses of methylprednisolone. J. clin. Invest. 52, 2629-2640 (1973). 6. Schulz G. and Beyer J. Grundlagen der antiphlogistischen und immunsuppressiven Therapie mit Corticosteroiden. Dt. med. Wschr. 103, 1925-1930 (1978). 7. Zimmermann B. Immunosuppression with anti-lymphocyte serum: A review of the effect of ALS on the immunoregulatory network. Aft. J. clin. exp. lmmun. 3, 171-182 (1982). 8. Heyworth M. F. Clinical experience with antilymphocyte serum. Immun. Rev. 65, 79 (1982). 9, Russel P. S., Colvin R. B. and Cosini. Monoclonal antibodies for the diagnosis and treatment of transplant rejection. A. Rev. Med. 35, 63 (1984). 10. Block L. H., Sutter P. M. and Mihatsch M. J. Cyclosporin A: Pharmacologic activity in the immune sytem and effects in clinical organ transplantation. Kiln. Wschr. 61, 1053-1062 (1983). I I. Dickneite G., Schorlemmer H. U., Walter P. and Sedlacek H. H. Graft survival in experimental transplantation could be prolonged by the action of the antitumoral drug 15-Deoxyspergualin. Transpl. Proc. 18 (1986) In press. 12. Sedlacek H. H., Weidmann E. and Seiler F. R. Tumor immunotherapy using Vibrio cholerae neuraminidase (VCN). In Bacteria and Cancer (Edited by Jeljaszewicz J., Pulverer G. and Roszowski W.), pp. 264-290. Academic Press, London (1982). 13. Latour Am6d6e. Le traitement des affections canc6reuses et tuberculeuses. Union m~d. de Paris; Gaz. M~d. de Paris, p. 8 (1850). 14. Busch W. Ober den EinfluB, welchen heftigere Erysipeln zuweilen auf organisierte Neubildungen ausiiben. Verh. naturh. Ver. d. preuss., Rheinl. u. Westphal., Bonn, 23, 28-30 (1866). 15. Adam Versuche zur Umstimmung eines K6rpers als Mittel zur Behandlung von Bazillentr/igern. Beitrag zur Klinik der Infektionskrankheiten und zur Immunitfitsforschung. Eppendorfer Festband 3, 1-2 (1914). 16, Much, Unabgestimmte Schutzimpfung. Dt. reed. Wschr. 26, (1919). 17. Buchner H. Zbl. Bakt. 8, 1 (1980). 18. Coley W. B. The treatment of malignant tumors by repeated inoculations of erysipelas, with a report of 10 original cases. Am. J. reed. Sci. 105, 487-511 (1893). 19. Calmette A. Ann. Inst. Pasteur 35, 561 (1921). 20. Buzello Die Grundlagen und praktische Anwendung der Proteintherapie. Dt reed. Wschr. 48 (1924). 21. Johnson A. G., Gaines S. and Landy M. Studies on the 0 antigen of Salmonella typhosa. J. exp. Med. 105, 225-246 (1956). 22. Math6 G. Immunoth6rapie active de la leuc6mie LI210 appliqu6e apr6s la geffe tumorale. Rev. franc. I~tud. clin. biol. 13, 881 (1968). 23. Math6 G., Amiel J. L., Schwarzenberg L., Schneider M., Cattan A., Schlumberger J. R., Hayat M. and De Vassal F. Active immunotherapy for acute lymphoblastic leukaemia. Lancet i, 697 (1969). 24. Halpern B., Prevot A. R., Biozzi G., Stiffel C., Mouton D., Morard J. C., Bouthilier Y. and Decreusefond C. Stimulation de l'activit6 phagocytaire du syst6me reticuloendothelial provoqu6 par Corynebact. parvum. J. Reticuloendothel. Soc. 1, 77 (1964). 25. Old L. J., Clarke D. A. and Benacerraf B. Effect of Bacillus Calmette-Gu6rin on transplanted tumors in the mouse. Nature 184, 291-292 (1959). 26. Math6 G., Pouillart P. and Lapeyrague F. Active immunotherapy of LI210 leukemia applied after the graft of tumour cells. Br. J. Cancer 23, 814 (1969). 27. Seiler F. R., Hofstaetter T., Kolar C., Kraemer H. P,, Schorlemmer H. U. and Sedlacek H. H. Immunommodulation: Immunstimulation und Immunsuppression (Edited by Erhardt und Ruschig). Arzneimittel: Entwicklung, Wirkung, Darstellung-Fortschritte 1972-1982, Verlag Chemic (1985).

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