Abstract
This article provides a brief history of the development of the field of immunotoxicology from one individual perspective and separates the discussion into five phases: the methods development and validation phase; the compound testing phase; the many meetings and organization of the specialty section phase; the mechanistic studies phase; and the guidelines phase. During the discussion of each phase, major highlights, accomplishments, contributors and key references are provided. The immunotoxicology guidelines for the testing of pharmaceutical chemical entities across the three major geographic axes are also presented and compared, along with some of the concerns raised by industry with these guidelines. The mandatory requirement of functional tests represents the major concern and difference between the Committee of Proprietary Medicinal Products (CPMP) and the new Food and Drug Administration (FDA) guidance. The scientific basis for the recommendation of the functional tests proposed in the guidance documents based on National Institute of Environmental Health and Safety/National Toxicology Program (NIEHS/NTP)-sponsored studies is described. Experience at Sanofi-Synthelabo, with the testing of 29 new chemical drug entities developed across a broad range of therapeutic classes using this testing paradigm and functional tests to define their immunotoxic potential, yielded a low number of compounds (6.8%) that produced any abnormal reaction. The two positive compounds might have been anticipated based on their pharmacology.
I want to thank The American College of Toxicology, its Council, and especially the Awards Committee for this recognition as the 2003 Distinguished Service Award recipient. This is a great personal honor for me for two important reasons. First, to be recognized by your peers for having contributed to the science of toxicology is a great privilege, especially because my training was in molecular biology, biochemistry, and immunology, although I have worked in toxicology for the past 25 years. Secondly, for the last 15 years, I have been employed in the pharmaceutical industry, involved in drug safety evaluation, and it is rare that someone in my industry receives such a prestigious award. Therefore, I sincerely thank the American College of Toxicology (ACT) and all of you who contributed to the achievements leading to this great honor. Please know that receiving this award means a great deal to me and is a symbol of the collaboration and help received from so many of you.
I would like to provide a brief history of the development of the field of immunotoxicology, which I will separate into five phases for purposes of discussion as follows: the methods development and validation phase; the compound testing phase; the many meetings and organization of the specialty section phase; the mechanistic studies phase; and the guidelines phase. Then I will describe the new immunotoxicology guidelines for the testing of pharmaceuticals across the three major geographic axes (Europe, the USA, and Japan).
As a point of reference, the immune system is considered to be a highly integrated network of cells that attracted the interest of toxicologists because these cells are known to be pivotal in host defense against infectious agents and neoplasia. These cells are derived from the hemopoietic pluripotent cells of the bone marrow that undergo differentiation through autocrine, lymphokine, chemokine, and endocrine control into the cells that comprise the mature immune system. The organization and interaction of the hemopoietic cells of the immune system is well described in the literature (Goldsby, Kindt, and Osborne 2000), as is the evidence that xenobiotics that can alter cell proliferation, differentiation, or activation can also potentially injure these cells, disrupt immune responses, and alter host resistance (for review, see Dean, House, and Luster 2001).
THE METHODS DEVELOPMENT AND VALIDATION PHASE AND MY INTRODUCTION
My first knowledge of the emerging field of immunotoxicology came in the Fall of 1997 while working in Bethesda, MD, when I was asked by Dr. Bob Weir to organize a 2-day symposium on immunotoxicology for the 1978 Gordon Research Conference (GRC) on Drug Safety. At that time, I was Director of Immunology and Cancer Biology at Litton Bionetics and the Project Director on several National Cancer Institute/National Institutes of Health (NIH) contracts studying cancer biology, the immune response against tumors, and the effect of anti-cancer drugs on the immune system in animals and humans. After trying repeatedly to explain to Dr. Weir that I know nothing about immunotoxicology and was not the person to organize this meeting, he said to me, “It is the research you are doing in your laboratory.” Armed with a strong desire to spend a week in New Hampshire, I went to the National Library of Medicine and did a literature search to find a recent review on the topic by Dr. Jeff Vos (1977) as well as several additional primary articles. This review was probably the first paper to define and establish this field and it stimulated my interest immensely. Dr. Vos had worked at National Institute of Environmental Health and Safety (NIEHS) as a visiting scientist with Dr. Jack Moore, who was the referee for the paper. I invited Dr. Moore to participate in the GRC because there were no funds to bring a scientist from Europe. Other workers in the emerging field of immunotoxicology who were invited to present included Drs. Loren Koller, Leland Loose, Mike Luster, A. Munson, and Raja Sharma. The meeting drew a great deal of interest from the drug safety community.
Shortly after the GRC, I published papers on a tiered-testing approach to study the effects of agents on the immune system, which was based on studies from my laboratory in Bethesda, with anticancer drugs (Dean et al. 1979a Dean et al. 1979b). This approach apparently caught the attention of Drs. Moore and Rawl at NIEHS, because they invited me to establish a laboratory of immunotoxicology in the Environmental Biology Branch shortly thereafter. The laboratory at NIEHS was established in July of 1979 and consisted of M. Luster, R. Lawson, L. Lauer, R. Luebke, and myself. Dr. Gary Boorman provided the immunopathology support and expertise from the National Toxicology Program (NTP).
Shortly after coming to NIEHS, I organized the first Workshop on Methods and Approaches for Assessing immunotoxicology in Williamsburg, VA (Fall 1979), that was attended by approximately 50 scientists and physicians. This Williamsburg meeting was immediately followed by an NIH Consensus Meeting at Research Triangle Park, NC, that defined specific research needs of this newly emerging field. This led to my first extramural assignment at NIEHS to develop a Request For Proposal and to award two contracts for the assessment, selection, and validation of immunotoxicological methods in rodents. These contracts were awarded in 1980 to Dr. Peter Thomas and the team at the Illinois Institute of Technology Research Institute (Chicago) and Drs. Page Morhan, Al Munson, and the staff at the Medical College of Virginia (Richmond). These contracts were probably among NIEHS’ most significant contribution in this area because, along with our intramural laboratory, they led to the selection, refinement, and validation of several immunological methods, and resulted in the training of numerous pre-and postdoctoral students.
The success of the program at NIEHS was due to Mike Luster’s knowledge of immunoassays and environmental chemicals, Gary Boorman’s expertise in pathology, and my background with immune function assays and host resistance models (Dean et al. 1981). During this period, we established the use of host resistance models as surrogates for immune alteration. Studies by the NIEHS team and our contract laboratories led to the demonstration that, by using only two or three assays of immune function, one could obtain a good correlation with changes in host resistance predicting biologically significant immune alterations in rodents (Luster et al. 1992).
Results from the interlaboratory selection and validation of immunotoxicology methods sponsored by the NTP, NIEHS, as described above, has yielded valuable information on the utility of individual tests and the optimum assay configuration to identify immunotoxic compounds in rodents. It also described a qualitative and quantitative relationship between immune tests and alterations in host resistance challenge models (Luster et al. 1992). In NTP-sponsored testing, the ability of each of the immune tests to predict an immunotoxic outcome was evaluated (Figure 1). The highest positive concordance was found with the T cell–dependent antibody plaque-forming cell (PFC) response to sheep erythrocytes (78%) and lymphocyte subpopulation surface marker expression analysis (83%) (Luster et al. 1992). Also, the ability of a combination of two or more tests in the panel to predict a positive outcome was examined, and the combination of PFC and natural killer cell cytotoxicity or cell surface phenotypic marker data increased the concordance to predict an immunotoxic compound by more than 90% (Figure 1). These data were pivotal in establishing the testing paradigms for immunosuppression described in most of the current immunotoxicology guidelines.
THE COMPOUND TESTING AND MECHANISTIC STUDIES PHASE
During the period at NIEHS, our group also studied and published on a number of important chemicals for potential effects on the immune system of the B6C3F1 mouse-asbestos, benzo(a)pyrene, cyclophosphamide, diethylstilbestrol, indomethacin, methyl and ethyl carbamate, mercuric chloride, orthophenylphenol phosphate, phorbol-12-myristate-13-acetate, Tris (2,3-dichloropropyl) phosphate, and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) (Boorman et al. 1980 Boorman et al. 1982; Dean et al. 1980, 1981, 1983; Dieter et al. 1983; Luster et al. 1980a, 1980b, 1981a, 1981b, 1982a, 1982b).
In 1982, Drs. Bob Neal and Jim Gibson invited me to join them at the Chemical Industry Institute of Toxicology (CIIT) to establish an immunotoxicology laboratory and molecular toxicology department. The decision was easy because the “Reagan Era” hiring freeze was still in place and the additional positions promised at NIEHS had not materialized. The 1980s, I think, represented the “golden years” of CIIT and this was a wonderful period in my career because of the excellent scientific staff with whom to exchange ideas and the outstanding quality of the pre-and postdoctoral students who came there for training. During this period in my own laboratory, I had the privilege of mentoring nine wonderfully gifted students (Table 1) who contributed vastly to the literature in immunotoxicology, and to an understanding of how chemicals alter the immune response. A number of significant papers were published during this period by my team at CIIT, as well as by other groups that helped to elucidate mechanisms of chemical induced injury to the immune system. Some of the key papers from our CIIT studies focused on the mechanism of immune effects induced by the following chemicals in mice: aflatoxin B-2; 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracened-iodihydrochloride; beta-hexa-chlorohexane (β-HCH); 1,3-butadiene; cyclosporin A; diethylstilbestrol; 7,12-dimethylbenz[a]anthracene (DMBA), and TCDD (Cornacoff et al. 1988; Dean et al. 1985a, 1986a, 1986b; Hastings, Tulis, and Dean 1988; House and Dean 1987; House et al. 1987, 1989a, 1989b, 1990; Pallardy, House, and Dean 1989; Selgrade et al. 1988; Thurmond et al. 1986, 1988, 1991; Ward et al. 1984). It was during the mid 1980s that our laboratory, and others, moved from just studying immune phenomena to exploring and understanding the mechanisms of chemical induced immune injury at a cellular and molecular level, which are summarized in several selected publications (Dean, House, and Luster 2001; Exon, Talcoss, and Koller 1985; Holsapple et al. 1996; Karol and Jin 1992; Kerkvliet et al. 1990; Pennicks et al. 1985; Tian and Lawrence 1996; White, Kawabata, and Ladics 1994; Vos 1984).
THE MANY MEETINGS AND ORGANIZATION OF THE SPECIALTY SECTION PHASE
Several key scientific meetings occurred during the 1980s in immunotoxicology (NIH Immunotoxicology Workshop, 1983; NATO Advanced Studies Institute on Immunotoxicology, 1984; WHO Meeting, 1984; International Seminar on the Immunological System as a Target for Toxic Damage, 1986; SGOMSEC 4 Workshop; Summer Schools in Immunotoxicology, and others) that demonstrated the importance of this area of toxicology to international academic, industry and regulatory agency scientists. Our team at NIEHS played a major role in organizing and participating in these meetings. Among the many meetings, one of the most pivotal meetings was organized (1984) by the World Health Organization (Alex Berlin) in Luxembourg and resulted in proceedings that provided the first formal definition of immunotoxicology (Berlin et al. 1987). This meeting brought together approximately 80 scientists from around the world to discuss appropriate methods and to review studies on several compounds. This meeting also allowed European and American scientists to realize the extent of mutual interest in this emerging field.
At the 1984 Society of Toxicology (SOT) annual meeting, Dr. Bob Dixon (president of SOT 1982–1983) requested that we initiate a petition to form a new specialty section for immunotoxicology in SOT. This process required the signatures of 50 SOT members who would support and join the new specialty section. My reward for this effort was that I was elected the founding President of the new Immunotoxicology Specialty Section (ITSS). Since its beginning in 1985, the ITSS has had substantial membership growth from 50 to now over 200 members and has contributed significantly to the scientific quality of the SOT annual meeting through symposiums, platform, and workshop sessions (i.e., five to seven per year), and high quality abstracts (i.e., >100 per year). Figure 2 is a picture showing some of these early pioneers of immunotoxicology gathered at the SOT Immunotoxicology Specialty Section meeting in 2001.
A Career Change
In 1988, I made a dramatic career change and joined the pharmaceutical industry to work for an old friend, Dr. Bob Dixon, then Vice President of Drug Safety at the Sterling-Winthrop Research Institute (Rensselaer, NY). I was attracted to the pharmaceutical industry because of Bob’s vision that the pharmaceutical industry should mimic what was done at NIEHS/NIH by pursuing science in the search of truth and “that we were not merely box-ticking when testing our drugs, but doing experiments to better understand their safety for man.” He fostered the view that through sound investigative studies, we could understand and explain the toxicities observed. The second attraction to the pharmaceutical industry was its strength in medicinal chemistry, which might allow one to “design out” the toxicity while trying to preserve the desired pharmacological activity. I was tremendously excited by both concepts and they have been our mantra during my 15 years in the pharmaceutical industry, although the former objective has been more easily accomplished that the latter. From the beginning, with Bob’s support, we established investigative laboratories in special pathology, molecular and cellular toxicology, and immunotoxicology. Unfortunately, Bob died of liver cancer a few months after I joined him at Sterling-Winthrop, but he will always be remembered as a great teacher, scientific leader, and friend to many whose contributions included founding the Target Organ Toxicity Symposium Series and the book series by the same name.
During my tenure in the pharmaceutical industry, I have survived three corporate mergers and participated in the registration of seven new drugs including three blockbusters—Plavix, Avapro, and Eloxatin. The pharmaceutical industry is an exciting, rewarding, and challenging place for a toxicologist to work if you can survive the anxiety created by the frequent corporate mergers.
During this period, our immunotoxicology laboratory at Sanofi-Synthelabo has studied four reference compounds (azathioprine, cyclosporine A, cyclophosphamide, and dexamethazone) as part of various interlaboratory validation studies, and 29 new chemical entities (NCEs) from many divergent pharmacological classes in our pipeline using the testing paradigm proposed in the Committee of Proprietary Medicinal Products (CPMP) guidance. What has been astounding to me is that from the varied array of pharmacological and chemical entities evaluated, we found that only 2 (6.8%) of the 29 NCEs produced any immunomodulatory effects. These results are described in detail in earlier publications (Dean, Hincks, and Remandet 1998; Dean and Remandet 1997).
THE GUIDELINES PHASE
Let’s now discuss some recent guideline-development activities for the immunotoxicology evaluation of pharmaceuticals. The interest of regulatory agencies in developing guidelines in this area is stimulated by the knowledge that some chemical and therapeutic drugs (i.e., primarily transplant rejection prevention and anticancer agents) have been shown to cause significant alterations of the immune system, and they want to prevent such agents from being introduced into widespread therapeutic use with such potential adverse effects. Because the safety of NCE is based on the weight of the evidence, this consideration would seem reasonable, given the fact that we consider multiple target organ effects in safety evaluation.
The safety testing of pharmaceuticals in Europe is regulated by the CPMP, which in October of 2002 published a Note for Guidance on Repeated Dose Toxicity (CPMP/SWP/1042/99), containing an appendix mandating specific immunotoxicology screening for pharmaceuticals (document available at http://www.emea.eu.int/pdfs/human/swp/104299en.pdf). Although the primary objective of this new guidance was to describe an overall approach for the safety testing of NCEs, it provided the first guidance mandating immunotoxicology screening of pharmaceutical NCEs. The appendix describes a tiered evaluation of NCEs and indicated that information gained in the standard toxicology evaluation should be used as a primary indicator for immunotoxicity. Emphasis in this document was placed on enhanced pathology and route hematology, and mandatory functional tests for the first time were incorporated in the initial screen and then extended as indicated. The three functional assays recommended (natural killer cell assay, surface marker determination, and assessment of the antibody response to a T-dependent antigen) were previously shown useful in predicting an immune alteration that correlated with changes in host resistance in studies performed and sponsored by the NTP (Luster et al. 1992). Industry raised concerns that required clarification of the new European Union (EU) immunotoxicology guidelines and resulted in a Drug Information Association (DIA)-sponsored workshop in Noordwijk, The Netherlands, in November 2001, which in turn led to a definitive publication (Putman et al. 2002) clarifying many of the issues with the EU guidelines.
At the DIA meeting, it was generally agreed that immunotoxicity screening should encompass hematology, lymphoid organ weights, and histology of lymphoid organs. It was recommended that histopathology follow parameters described by Kuper et al. (2000) to increase consistency and sensitivity. The majority of the participants supported the view that disturbance of the functionality of the immune system may not always be detected by histopathology alone and suggested that some functional measures were required. It was the consensus that challenge with a T-dependent antigen is likely the best stand-alone assay to detect functional impairment of the immune system in rodents. The major clarification from this meeting was that complete immune assessment was not required prior to the start of clinical phase I, but it was recommended during phase II and before the start of phase III, which will significantly reduce the number of NCEs requiring study. The long-running debate is whether the standard pathology done in the repeated-dose study is adequate alone to detect an immunotoxic effect, or if a functional test is required.
In the United States, our Center for Drug Evaluation and Research, Food and Drug Administration (CDER/FDA), released a guidance document in October of 2002 entitled Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs (document available at http://www.fda.gov/cder/guidance/4945fnl.pdf). The approach of this very comprehensive document was to describe a menu of potential adverse events, including immunosuppression, immunogenicity, hypersensitivity, autoimmunity, and adverse immunostimulation, that could result from exposure to NCEs and that should be considered in any safety-testing paradigm. The FDA guidance document describes each of the five types of immune effects in detail and provides approaches and suggested methodologies to study each of them (Hastings 2002). A decision tree was presented to assist with determining if additional studies were required (Figure 3). The FDA document is similar to the CPMP document in that it states that results from the repeated-dose safety studies should provide some preliminary evidence of an immune effect, but differs from the CPMP guidance in that functional tests are not mandated, and further studies are determined on a case-by-case approach.
Regulatory guidance in Japan has been slower in developing. In 1999, the Japanese Pharmaceutical Manufacturers Association (JPMA) published two documents: International Trends in Immunotoxicity Studies of Medicinal Products, and Survey on Antigenicity and Immunotoxicity Studies of Medicinal Products. These two documents are merely a survey of immunotoxicology methods and study designs used in Japan, and do not mandate any testing requirements of NCEs. At the DIA workshop, Dr. K. Nakamura, representing the JPMA, presented an Interim Draft Guidance of Immunotoxicity Testing that described the current thinking of the pharmaceutical industry and the regulatory authority in Japan, but this guidance still has not been released. Table 2 compares and contrasts these different geographic guidance approaches for the immunotoxicity testing of pharmaceutical NCEs.
During the ICH VI Meeting this past November in Japan, guidance on immunotoxicology testing of pharmaceuticals was accepted as a new harmonization topic (S8) by the International Committee on Harminization (ICH) Steering Committee. Thus, over the next few years, we should expect to see a harmonized guideline for the immunotoxicity evaluation of NCEs. My experience from past ICH harmonization exercises is that often the most rigorous standard is the accepted position. Until a harmonized guideline is achieved, Sanofi-Synthelabo, like many other international pharmaceutical companies, will continue to work in accordance with the more rigorous EU guideline because we register our drugs internationally, in parallel, and we cannot afford a delay due to an incomplete file or inadequate safety data. It is also our view, and, I believe, supported by the literature, that pathology alone, while adequate to identify many potential immuntoxic agents, cannot with 100% confidence identify all immunologically active agents that might be identified in one or more functional screening tests. This suggestion would not be a surprise to a student of immunology or the immunotoxicology literature.
In closing, I want to thank the ACT once again for this prestigious award, and especially thank my lovely wife and longtime friend Suellen, who has spent many evenings alone while I traveled in pursuit of my second love—the science of toxicology.