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THE SOCIETY OF TOXICOLOGY CONTEMPORARY CONCEPTS IN TOXICOLOGY SECTION U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES FOOD AND DRUG ADMINISTRATION OFFICE OF WOMEN'S HEALTH CENTER FOR BIOLOGICS EVALUATION AND RESEARCH Arlington, Virginia Monday, December 2, 2002 [Transcript Prepared From A Tape Recording.] CONTENTS MORNING SESSION: WELCOME KAREN MIDTHUN, M.D. Director, Office of Vaccines Research & Review, CBER/FDA NON-CLINICAL SAFETY ASSESSMENT OF PREVENTIVE VACCINES PRESENTATIONS: [Note: All presentations were accompanied by slides] FDA PERSPECTIVE: ELIZABETH M. SUTKOWSKI, Ph.D. Office of Vaccines Research & Review, FDA INDUSTRY PERSPECTIVE: FRANCOIS VERDIER, Pharm.D., Ph.D., Product Safety Assessment, Aventis Pasteur ROUNDTABLE DISCUSSION: SCIENTIFIC AND TECHNICAL CHALLENGES IN PRECLINICAL SAFETY TESTING OF VACCINES SALLY HARGUS, Ph.D., CBER, FDA, Moderator TOPIC ONE: "The Relevance of Animal Studies for Non-Clinical Safety Evaluation of Vaccines" BRIAN LEDWITH, Ph.D., Director, Biologic Safety Assessment, Merck TOPIC TWO: "Applicability of Traditional Drug Toxicity Study Designs for Safety Evaluation of Vaccines" GARVIN WARNER, Ph.D., Director, Exploratory Drug Safety, Wyeth Research * * * AFTERNOON SESSION: ROUNDTABLE DISCUSSION [Cont'd.]: TOPIC THREE: "Animal Models Appropriate for Assessing Immunotoxicology and Immunopathology" MICHAEL LUSTER, Ph.D., Chief, Toxicology & Molecular Biology Branch, CDC, NIOSH TOPIC FOUR: "Safety Evaluation of Adjuvants: Should They Be Considered Separately, or Only in the Context of the Final Vaccine?" NATALIE GARCON, Pharm.D., Ph.D., GlaxoSmithKline ROUNDTABLE DISCUSSION: AUTOIMMUNITY-HYPERSENSITIVITY AND VACCINE SAFETY ASSESSMENT TOPIC ONE: "Status of Non-Clinical Methods for Autoimmunity" PAUL-HENRI LAMBERT, M.D., Chief, Vaccine Research & Development, WHO TOPIC TWO: "Status of Non-Clinical Methods for Hypersensitivity" FRANCOIS VERDIER, Pharm.D., Ph.D., Aventis Pasteur QUESTION & ANSWER SESSION PROCEEDINGS DR. MIDTHUN: Would everyone please take their seats? Good morning, and welcome. It gives me great pleasure to welcome all of you to this Workshop on Non-Clinical Safety Evaluation of Preventive Vaccines. I'd like to start by thanking our co-sponsors--in particular, the Society of Toxicology and the Contemporary Concepts in Toxicology Section--for their help in organizing and contributing to this workshop. And a special thanks to Shawn Lamb, the executive director of the SOT, and her staff, for their extremely wonderful help in putting all of this together. We'd also like to thank, and very much appreciate the support extended by the FDA Office of Women's Health, and for their significant contribution to the funding for this workshop. And I'd also like to give a special thanks to the staff of CBER in pulling all of this together. I'd also like to acknowledge the organizing committee members. A special thanks to Marion Gruber and Liz Sutkowski, from the Office of Vaccines, for working extremely hard to develop and coordinate the workshop. Marion has been working for several years to develop the Office of Vaccines' policy on preclinical toxicity testing; and in particular, to develop reproductive toxicology testing. And Elizabeth Sutkowski is the chair of the preclinical toxicity testing working group within CBER, and is drafting a guidance for industry on preclinical toxicity testing of preventive vaccines. We'd also like to thank Mercedes Serabian, from the Office of Therapeutics, and Sally Hargus, from the Office of Vaccines, for their help in organizing this workshop, and for acting as moderators for the roundtable discussions. We also thank Christine Everett for her help in ushering through the co-sponsorship agreement and the approval of the funding from the Office of Women's Health. We gratefully acknowledge Francois Verdier, for his help in organizing the workshop; and in particular for his recommending that this first day of the workshop be dedicated to discuss preclinical safety of testing of vaccines in general. We also gratefully acknowledge Dr. Kenneth Hastings of the Center for Drugs, for his help in coordinating the workshop; and in particular, for his recommendations for speakers for the workshop. And we also thank Robert House, of DynPort Vaccine Company, for his help in organizing the workshop; and in particular, for securing the approval of the co-sponsorship for the CCT section of the SOT. This morning I'd like to discuss the key components of the safety evaluation of biologics; which, of course, include preventive vaccines. I'm going to briefly review approaches to toxicity assessments of preventive vaccines, past and present. I'll touch on current challenges and issues related to non-clinical safety assessment of vaccines. And I'll describe some initiatives addressing non-clinical safety assessments of vaccines, and how the regulatory process is evolving in this area. Clearly, assuring the safety of biologics is at the forefront of CBER's mission. What are some key components of the safety assessment? The safety assessment of preventive vaccines is a continuous process that begins with the development of a vaccine candidate at the pre-IND stage. It continues through the various stages of clinical development during the IND phase. And it continues onward after licensure, as well, with post-marketing surveillance, and also with inspections that are ongoing at manufacturing sites. It includes the characterization of the product by physical, chemical, and biological testing. It includes an adequate control of the manufacturing process, and the development and establishment of adequate lot release tests to assure the safety, purity, and potency of the products. It also includes toxicity assessments in animals, clinical safety assessments and, again, surveillance after licensure, as well. Over the next couple of days, we'll be discussing the non-clinical safety assessment of a product focusing on animal safety testing prior to introducing the product into the clinic and any further safety evaluation of animals that may be needed to be performed during and in parallel with clinical development of the product. How is "safety" defined? Well, in the Code of Federal Regulations it states that "safety" is "relative freedom from harmful effect to persons affected directly or indirectly by a product when prudently administered, taking into consideration the character of the product in relation to the condition of the recipient at the time." Thus, given the diversity of preventive vaccine products, the safety evaluation needs to consider the character of the product, the methods of manufacture, and the indications. It's critical that early in product development agreement is reached between the Center for Biologics and a vaccine developer, to assure that methods and standards for the preclinical and clinical safety evaluation of a product are adequate. Historically, the non-clinical safety assessment for preventive vaccines has often not included toxicity studies in animal models. This is because vaccines have not been viewed as inherently toxic, and vaccines are generally administered in limited dosages over months or even years. However, progress in the field of biotechnology has accelerated the development of a broad range of novel vaccines, and the composition of vaccine products has evolved from attenuated or inactivated whole-cell organisms, to protein polysaccharide conjugates, peptides, recombinant proteins, DNA vaccines, etcetera. More recently, there has been a generation of a wide range of complex vaccine products and vaccine technologies that are often combined with novel adjuvants, administered in new delivery systems, and administered by new routes of administration. These advances have resulted in an increased focus on non-clinical safety assessment of these products and whether there is a need for initial phase-one clinical studies to be supported by preclinical toxicity data in animals. In contrast to most drugs and biological products that are predominantly developed to treat ill patients, vaccines primarily are given to large numbers of healthy people, oftentimes predominantly healthy infants and children. And this places significant emphasis on their safety. Also, for several vaccines the incidence of the infectious diseases that they are intended to prevent is quite low. Therefore, a high percentage of vaccinated people will never be exposed to the infectious agent. Thus, the benefit of the vaccine in preventing the infectious disease may be difficult to appreciate at the individual level. Thus, there is low tolerance for significant adverse events associated with vaccines--that is, caused by vaccines. Given these findings, and the context of novel vaccine development, there is an increased focus on the safety assessment in animal models. If the preclinical safety assessment is deemed to be insufficient, this can lead to a clinical hold for an IND. The Code of Federal Regulations states that an IND should include data from pharmacologic and toxicologic studies that allow the sponsor to conclude that it is reasonably safe to conduct a proposed clinical investigation. The main challenge in establishing a predictive non-clinical safety assessment comes from the fact that vaccines act through complex multi-stage mechanisms. Thus, the detection of toxicity for vaccines is likely to be more complex than for conventional chemically-derived drug products, because safety concerns may result from the immune response to the vaccine. Thus, toxicity testing programs recommended for conventional drug products may not always be applicable to vaccine products. The non-clinical safety assessment of vaccines represents a new and evolving field. And clearly, consensus is needed among industry, academia, and regulatory authorities regarding the most appropriate and scientifically sound approaches to this area. And there are a number of questions to address: For which products should toxicity testing be performed? What are the criteria for selecting the appropriate route of administration, doses, and schedule? How should the toxicity of adjuvants be evaluated? What animal models should be used? And how should one incorporate alternative methods into non-clinical safety assessments? Depending on the target population and vaccine indication, it may be necessary to conduct special non-clinical safety assessments. In particular, if a target population for the product includes pregnant women or females of reproductive age, reproductive toxicity studies should be considered. We have dedicated the second day of this workshop to address this important subject. FDA announced in the Federal Register in September 2000 the availability of a draft document entitled "Guidance for Industry, Considerations for Reproductive Toxicity Studies for Preventive Vaccines for Infectious Disease Indications," providing information to sponsors regarding assessments of the reproductive toxicity potential for preventive vaccines indicated for maternal immunization and females of reproductive age. Industry has provided comments on this document and, because of the complexity of the issues and the concerns raised, we decided to discuss these in a public forum among experts in the field. Thus, tomorrow we will address technical aspects, experimental design, and animal models for developmental toxicity studies, in order to reach a consensus on how to best perform developmental toxicity studies for preventive vaccines, and the type of information that can be derived from such studies, to assure that it will be relevant and useful for assessment of human risk. A number of working groups have been established at CBER, not only to address the non-clinical safety assessment, but a number of other aspects of safety. And I'll just mention these briefly, although they're not the focus today. For example, looking at the safety aspects of DNA vaccines, the cell substrates used to manufacture vaccines, and also keeping abreast of the best ways to test for adventitious agents. There is a CBER reviewer document, as CBER has been engaged in the process of developing guidance for the preclinical toxicity testing of preventive vaccines. The internal reviewer document is entitled "Preclinical Toxicity Studies for Vaccines To Support Initiation of Clinical Studies." And that's an internal reviewer document. And Dr. Sutkowski will discuss this with you in her presentation. This document will eventually form the basis for a guidance for industry document for non-clinical safety evaluation of vaccines. The goal is to publish a document that is specifically tailored to preclinical and non-clinical safety assessment of preventive vaccines. And that, besides a discussion of general toxicity assessments, includes special considerations for individual product categories, adjuvantive vaccines and other routes of administration. Issues pertaining to the guidance document on reproductive toxicity studies will be presented by Dr. Gruber, and will be discussed tomorrow. How is the regulatory process evolving? Well, toxicity assessments will be a part of the product characterization for certain vaccines. CBER will continue to use a scientifically based, case-by-case approach to toxicity assessment. In summary, non-clinical toxicity assessment is a key component in the development of preventive vaccines. The challenge in predictive safety assessments for preventive vaccines is due to the fact that vaccines are not conventional drugs. We need to discuss and reach consensus on scientific and technical approaches for toxicity assessments that are specific for vaccines. These approaches should be optimized to lead to the generation of interpretable data, the wise use of animal resources; and should facilitate the development of safe products, not delay product development. It gives me great pleasure to introduce our next speaker. And that's Dr. Liz Sutkowski. She will be presenting the FDA perspective of the non-clinical safety assessment of preventive vaccines. Dr. Sutkowski is a scientific reviewer in DVRPA--in the Division of Vaccines and Related Products Applications, in the Office of Vaccines--and has chaired the working group on the preclinical safety testing of preventive vaccines. She has a wealth of experience that she brings with her: a background in biochemistry; post-doctoral work in the departments of pharmacology at Georgetown University and the University of Washington. She also had many years of experience in the division of cytokine biology in the Center for Biologics, before coming to the Office of Vaccines. So it gives me great pleasure to introduce Dr. Liz Sutkowski. [Applause.] FDA PERSPECTIVE: BY ELIZABETH M. SUTKOWSKI, PH.D., OFFICE OF VACCINES RESEARCH & REVIEW, FDA DR. SUTKOWSKI: Thank you, Dr. Midthun, for giving an overview of the initiatives that are ongoing in our office on the non-clinical safety evaluation programs that we have and our addressing on this evolving field. I'll be giving the FDA perspective today on non-clinical safety assessments of preventive vaccines regulated by CBER. As Dr. Midthun mentioned, the Office of Vaccines is giving consideration to whether or not, prior to proceeding into phase I clinical trials, there is going to be extra consideration given to whether or not non-clinical safety assessments will need to be supported by toxicity testing in animals. And so the purpose we have gathered here today is to discuss this evolving field, and to facilitate discussion between regulators and researchers in the fields of immunology and toxicology, and to address specific questions that we have in our minds for generating the guidance that Dr. Midthun mentioned. The objective of my talk today in introducing this workshop is to just go over the challenges that we are facing in toxicity assessments for preventive vaccines, and to go over how the regulatory process is evolving within CBER, and then to go over the current approach that we are taking to toxicity assessments for preventive vaccines; with the idea that the approach we have is evolving, and is not written in stone, and we are here to seek input from all of you. Today I just want to focus on preventive vaccines, and say that our office regulates preventive vaccines and therapeutic vaccines for infectious disease indications. So we do not regulate other therapeutic vaccines, such as cancer vaccines. And I wanted to just make sure that everybody is on the same page, and provide a definition of "preventive vaccine." And I'll just give you a few minutes to read that. Then another couple of other items I wanted to make sure that we had the same perspective on, in terms of definitions, were the preclinical safety assessments. We feel this includes the product characterization, as related to safety, animal safety testing. And both of those things are required for initiating clinical trials. And preclinical safety assessment, then, is a subset of non-clinical safety assessment; which would include, in addition to preclinical safety, any further safety assessments that would be required during the various stages of clinical or product development. Such as, if any significant changes are made to the product and/or the formulation, then there may be additional safety studies required; and/or if any safety concerns arise during the phase I or phase two clinical trials, then additional safety studies may be required. This slide lists the key components in non-clinical studies for preventive vaccines. On the left you see there is product characterization, in terms of characterizing the product by biological, physical, and chemical means. And then the next important aspect is manufacturing and the challenge in developing a manufacturing process that, as the product development proceeds, begins with first having control over the starting materials, and then gaining in process control testing and, as the development proceeds to phase three, to establish validated process procedures and to ensure consistency in manufacture by establishing lot release specifications that ensure product purity and potency, and to fully evaluate the stability. But for today's purposes, we'll be focusing on the right-hand side of the slide. And this includes safety studies that can be performed either in vitro, or animal studies that would include immunogenicity. And this might be part of establishing the potency of the product. It would also include pyrogenicity testing, which would be performed as part of the purity analysis of the product. And also, of course, general safety testing. And for certain types of products, there may need to be some neurovirulence testing performed--for example, for live or attenuated organisms. And then, for possibly attenuated organisms or inactivated toxins, you might look for reversion to virulence for those types of products. And in addition, there may be a need to do some additional safety studies. And this could include a GLP--or "good laboratory practices"--compliant toxicity study in animals. Okay. Dr. Midthun mentioned that vaccines have been generally thought to be inherently safe products. But there is precedence for CBER requesting toxicity studies for vaccines. For example, when the target population includes pregnant women; when there is either a new route of administration or the product contains a novel adjuvant; and also, as I mentioned, when there are some adverse effects that may be observed in the clinical trials; then the sponsor may be asked to examine potential toxicity of the vaccine in additional safety studies designed to replicate the specific clinical event. And if you were to look in the currently available guidance for principles on designing toxicity studies, you could look at these available guidance documents. The first one is the CPMP note for guidance--and this is a very comprehensive document--on designing preclinical pharmacological and toxicological testing for vaccines. And then, there is the ICH S6 document, which focuses on biotechnology-derived pharmaceuticals; and the ICH S5a document, which describes toxicity testing for effects on reproduction. And CBER has referred to this document in their own. The next one is the draft guidance document that CBER has published in September of 2000. And in the CBER document, CBER elaborated more on the considerations for reproductive toxicity studies. And finally, there is the EMEA concept paper on the development of the CPMP note for guidance on requirements for the evaluation of new adjuvants in vaccines. And there are also several published articles on designing toxicity studies for vaccines containing adjuvants; one of which is one published by Doctors Goldenthal, Joy Cavagnaro, Carl Alving, and Fred Vogel. And we at the Office of Vaccines use this article a lot to help design clinical studies, non-clinical safety assessment studies for vaccines containing adjuvants, and for adjuvants. Given the availability of these guidance documents, we feel that there still are uncertainties regarding the toxicity assessments of vaccines, such as those listed on this slide. For example, there is still uncertainty regarding which of the documents are most applicable for use for developing toxicity studies for preventive vaccines regulated by CBER, and whether or not toxicity testing should always be part of the product development. Is it necessary for every type of product? And if it is required, during what phase of clinical development should the studies be done? And finally, if a study is needed, should it be designed using the conventional toxicity testing approach used for drugs; and whether or not that would be applicable for vaccines. So one of the reasons that it's difficult to write a vaccine-specific document, and probably why it hasn't been done so far, is that vaccines are a very complex, diverse class of biologic products. And it's difficult to come up with an appropriate study design, because vaccines act through a very complex mechanism, whereby the product itself is not the final triggering component; but instead, it's the elements of the immune system that are the effectors. And so some of the questions that one has to address in designing toxicity studies for preventive vaccines is to try to approach all of these issues at the same time, and to design studies that look for inherent toxicity of the vaccine, as well as toxicity of the impurities and contaminants, as well as any toxicity that may be due to the components and individual antigens and other components interacting, and the toxicity linked to the immune response induced by the antigen. So in terms of telling you today how the regulatory process is evolving, as Dr. Midthun mentioned, we feel that there is a framework needed for non-clinical safety testing for preventive vaccines. And we plan to use the existing documents as a base to develop the guidance. And the goals of the working group are to make regulatory recommendations and/or requirements more transparent. And we hope that the guidance document would facilitate discussions between regulatory agencies and sponsors and promote relevant and consistent non-clinical testing and review within CBER. And so, as Dr. Midthun mentioned, we have formed a preclinical safety testing and preventive vaccines working group. And we have already written the first guidance that we plan to write, which is the CBER reviewer internal document entitled, "Preclinical Toxicity Studies for Vaccines To Support Initiation of Clinical Studies." Okay. I just wanted to mention that the CBER internal reviewer document is going to form the basis for the next document that we plan to write, which is a stand-alone guidance document for guidance for industry, entitled, "Non-Clinical Safety Evaluation of Preventive Vaccines." So we plan to use the CBER reviewer internal document as the basis to describe the general approach to toxicity testing in novel vaccines. And that would be the basis for the document. And then in addition, we would have sections on the individual product categories, such as those listed here, and also combination vaccines, and adjuvanted vaccines, and products given by novel routes. So that is one we are still working on. And one reason why we are here today is to get input on the issues, so we can continue to work on that document. And now I'd just like to go over what principles we have listed in our CBER reviewer document, so you are aware of our approach so far, although it's not written in stone. This is what it is to date: We have tried to clarify for what product types preclinical toxicity assessment is needed; We have tried to clarify the timing, the extent, and the approaches to the design of the safety studies, to support initiation of clinical trials; And we have described the extent of preclinical documentation required prior to initiating the clinical trials. So the principles that we have outlined in the document indicate that there is a need for preclinical toxicity--or that the need will depend on risk-benefit considerations, what the target population is, what the route of administration is. And we will also need to look at the available clinical data from the use of related products. And you'll also need to consider product features, such as novelty. And finally, the availability of animal models, relevant animal models. And the bottom line is that, in considering all of these different items, one needs to use scientific judgment, and that should be the basis for the decision. And it will be on a case-by-case basis. And this is just a slide to illustrate sort of the clearer areas where you can decide whether or not a preclinical toxicity study would be needed for a particular kind of product. It would likely be needed for a product if it contains a novel adjuvant or a toxic adjuvant for which there is no existing preclinical or clinical data; and if the product is from a novel product class for which we don't have extensive clinical experience; or if it's to be given by a novel route of administration. And it's likely that you may not need a toxicity study to go into a phase I study if the product category is one from which we have extensive clinical experience, or a product for which there is a great amount of product characterization. And this would usually include already licensed products. And also, combination products, including licensed products, you would likely not need to do a toxicity study again. In terms of the timing, the CBER reviewer document indicates that if the product is one for which the toxicity study is going to have to be done, then it should be done prior to initiating the phase I clinical trials. And we recommend in the reviewer document that the sponsors agree with CBER, prior to or during the pre-IND meeting, in terms of the design of the preclinical toxicity study. And this would, of course, require having adequate information from the sponsor on the clinical plan they propose. And we also recommend to reviewers that the sponsors submit the protocols to us for review prior to initiating the animal studies. And once the toxicity studies have been done and you come in with the original submission, the sponsor should include the toxicity study report, which should include a full tabulation of data and line listings, all organized into well organized tables. And finally, the additional toxicity studies, in addition to those required to phase I, may be necessary as the product and clinical development continues. And so the next part of the CBER reviewer document, and of course the meeting today, is to focus on this question: How to design appropriate non-clinical safety evaluation programs for preventive vaccines. I would like to go over the considerations that we have outlined in our CBER reviewer document, in terms of what things to consider for designing the toxicity study. And the goals of the toxicity study should be adequate to identify and characterize toxic effects. And we understand that no one study design is perfect for all product categories. But in general, the parameters to be considered in designing the toxicity study should consider animal species and strain; and the clinical plan, in terms of what's the proposed dosage form, dose, and route of exposure, and frequency of exposure, and whether or not the product will be delivered by any particular kind of device; and then of course, the product features, in terms of whether it's novel; and other product features and previous data that may need to be considered in designing the appropriate toxicity study, in terms of what is already known about the product. And finally, the toxicity study should be designed to try to evaluate potential toxic effects on the target organs and the immune system. And the reversibility of any observed toxic effects should be evaluated. So in terms of the approach that we currently have in our CBER reviewer document, the principles are outlined here. But once again, it's not carved in stone. We would recommend that sponsors could either do a dedicated stand-alone toxicity study, or they could do the toxicity study in combination with other safety, activity, and efficacy studies that they would be planning to do. We feel it's very important to use the relevant vaccine formulation. For example, if the product will be containing a novel adjuvant, you would need to look at the adjuvant alone and in the formulation that is planned for clinical use. And in terms of correlating with the clinical study, you also need to use the route of administration and the dose that you plan to use in the clinical study. And the total number of doses should exceed the number of clinically administered doses. And when giving the doses to the animals, it should be done episodically. And the toxicity study should include control arms, appropriate control arms. And finally, the study should be done in a relevant animal model. Now, just a few words on selecting a relevant animal model. The animal model should be chosen to evaluate safety in an animal model that mounts an immune response to the vaccine and, if possible, an immune response that's predictive for the human response. And additional considerations for choosing the animal model might include the age of the animal relative to the clinical study that's planned; for example, whether the study will be done in the elderly, or in the pediatric population. Another consideration for choosing the animal model is whether or not to use naive animals, versus partially immune or immune animals. So in our CBER reviewer document, this slide sort of outlines the parameters that we recommend be monitored. The study should look for local reactogenic and systemic events and immune mediated events; and should also include in-life parameters, such as clinical observations, body weight, and food consumption; and also, laboratory parameters, serum chemistries, hematology, and immunogenicity. And full necropsy should be performed, to include evaluation of organ description, weights. And this should also include selected histopathology. And finally, histopathology on immune system target organs should be performed, as well as immunogenicity in the laboratory parameters. So in summary, I think I have told you that non-clinical safety assessment, we feel in OVRR, is a key component in vaccine development; and that we are developing vaccine-specific guidance for non-clinical safety assessment of vaccines; and that the approaches towards toxicity testing for certain products we have tried to define, but that's still open for discussion. And so we basically wanted to answer two questions: For which product category type should toxicity testing be performed? And, how to best design appropriate toxicity tests for preventive vaccines. Just to go over now what we're here for today and what we hope to accomplish, we plan to discuss, and would like to invite you to discuss, the methodologies to determine potential adverse effects of new vaccines and adjuvants. We plan to discuss toxicity study designs and animal models, relevant animal models. And if possible, we would like to try to reach a consensus on the most appropriate--that is, the most scientifically sound--yet feasible approach to safety assessment of investigational new vaccine products. We would like to consider all of these aspects. And we have provided some questions in your packets. And we hope to deal with all of these topics today, and we really are seeking your input. And then, that pretty much does it for my presentation. If there are any questions regarding clarification of what we are trying to accomplish today, I'll take those questions; but otherwise, I think I'll hold any questions on the topics till the roundtable discussions. If there are no further questions, then I would-- PARTICIPANT [In Audience]: Liz? DR. SUTKOWSKI: Yes? PARTICIPANT [In Audience]: You talked about product class, and I'm not sure what you mean by that. DR. SUTKOWSKI: I guess I should have said "product category type." You know, is it a DNA vaccine? Is it a live organism? Is it any of those products that I had listed there, product types? Does it have an adjuvant? PARTICIPANT [In Audience]: There are some product categories that do not [inaudible] safety testing [inaudible]. DR. SUTKOWSKI: Well, the slide where I have where it's likely, no. Those are generally the kind of product categories that it's not required. But that's just a product category type. Yes? PARTICIPANT [In Audience]: Do you have a time line for turning your internal document into a formal guidance document? DR. SUTKOWSKI: We're hoping to do that in the next year. [No Further Questions.] DR. SUTKOWSKI: Okay, then. Thank you. [Applause.] DR. SUTKOWSKI: Now it gives me great pleasure to introduce the next speaker, Dr. Francois Verdier. Dr. Francois Verdier, PharmD, PhD, is the head of product safety assessment at Aventis Pasteur. He is in charge of establishing and assessing the non-clinical safety investigations required for new vaccines and adjuvant for clinical trials and marketing submissions. He is also involved in the safety issues for commercialized vaccines. He worked previously for a contract research organization, first managing toxicology studies, and then advising pharmaceutical companies on the toxicology requirements for pharmaceuticals, and particularly for biotechnology-derived products. He graduated in pharmacy at the University of Lyons, and received his PhD in immunotoxicology in the University of Paris. And this is still one of his fields of expertise. Francois Verdier is also a Eurotox-registered toxicologist, and a French national expert for the OECD guidelines. Francois, thank you for coming today. INDUSTRY PERSPECTIVE: BY FRANCOIS VERDIER, PHARM.D., PH.D., PRODUCT SAFETY ASSESSMENT, AVENTIS PASTEUR DR. VERDIER: Thank you, Elizabeth, for this kind introduction. So thank you, too, also, all FDA and SOT members, for the organization of this meeting. I think it's a great opportunity to discuss vaccine safety and to make progress in vaccine development. In my presentation, I would like to present the industry perspective. And you will see that there are a lot of overlaps with Elizabeth's presentation. So it means that we have a lot of agreement with the FDA regarding vaccine safety assessment. Also, I would like to mention that I did not make any survey in the vaccine industry to prepare my presentation, so this is my position. And I hope that during the discussion session I will have some challenges from my colleagues from other vaccine companies, in order to have a fruitful meeting with fruitful discussion. First, as an introduction, I would like to mention some trends concerning perceived vaccine safety. It was already touched on by the first speaker, and I would like to reinforce the fact that it's true that vaccines provide undisputed benefits to human health. But also, vaccine safety becomes a major public concern, particularly in developed countries. It is true that the majority of vaccines are given to healthy children. And it is also true that the risk-benefits ratio is looked at on the individual level. People expect a risk of zero from vaccine, even if it is theoretically impossible. And we know that perception of risk outweighs the perception of benefit in the public. And we can see also an increase in the activity of anti-vaccine groups. So taking into account these trends, and also perhaps due to some recent public health issues, such as the "Mad Cow" Disease, or the contaminated food product issue, there is an increased responsibility for the agencies and also for the vaccine manufacturers to work on vaccine safety assessment and to develop new methods in this way. I would like to illustrate these trends--at least hear some unsubstantiated claims between vaccines and disease. Perhaps for the two first claims there are some scientific hypotheses: Lyme vaccine and autoimmune arthritis, with the possibility of molecular mimicry in terms of vaccine; and Guillain-Barre syndrome. But for the last four examples, there are no real scientific data explaining these hypotheses: Combined vaccines and autoimmune diabetes; Hep-B and multiple sclerosis, this is mainly a French issue; MMR vaccine and autism, that is mainly a U.K. issue; and recently, aluminum hydroxide on macrophagic myofascitis, again mainly located in France. So at least from these claims it's obvious that we need to provide good scientific data, good non-clinical safety data, to argue on these claims. Also, if you are not yet convinced about the usefulness of non-clinical and clinical safety studies, I have listed here some examples of adverse reactions observed during non-clinical studies or during clinical studies. And it could be tissue necrosis at the injection site during animal studies. Kidney lesions, also: I observed this kind of lesion in primates after the administration of a cancer vaccine with GM-CSF as an adjuvant. Also, a vaccine antibody binding to animal tissues. And we observed that with polysaccharide Meninges-B vaccine. However, this binding was not associated with adverse reaction. During clinical studies, I have listed here some adverse reactions. The old but very bad story of the Formalin inactivated RSV vaccine. [Tape Change.] DR. VERDIER: Also, I noted some fever after the administration of a Japanese encephalitis virus vaccine during a clinical trial. And also--less severe again--swelling after repeated administration of cellular pertussis vaccine. So in front of these findings there are two potential strategies, two potential positions: either what I call the "ostrich strategy," or the "hunting dog strategy." And you will see at the end of my presentation which is for me the best one. I would like also to reinforce one of the slides from Elizabeth; the fact that vaccine safety evaluation is not limited to toxicity studies, as for a lot of biotech drugs. It's clear that we have to take into account the data provided by our colleagues from the quality control department. It is very important to know the quality of the raw materials; to know the stability of the product, including the genetic stability for viral construct. Elizabeth mentioned also some biological assays, such as general safety tests, neurovirulence, replication competency for viral vector. And all these data should be evaluated with the non-clinical safety studies to build the preclinical package for the safety of the product. Also, it was mentioned this morning that it's quite difficult for toxicologists to work on vaccine, because there are in fact various kinds of toxic effects which need to be taken into account. The first one, intrinsic toxicity, is perhaps not the major one with vaccines, because we are giving low quantity of antigens and the frequency of administration is not so high. But it could be applicable to adjuvant or excipient mixed with the antigens. The other type of toxicity is the toxicity associated with the pharmacodynamic activity of the vaccine, and it's probably more important for vaccine; for example, the cross reactivity between self antigens and vaccine-produced antibodies. It could be also the modification of the TH1/TH2 orientation, and any other potential toxic effect associated with the immune response triggered by the vaccine. More complex is what I have called the biological toxicity; namely, the adverse responses that are related to the activation of preexisting biological processes. And this is, for example, the exacerbation of preexisting autoimmune diabetes. Also, we have to keep in mind the potential adverse reaction due to the interaction either between different antigens or between the antigen and the potential adjuvant. And last but not least, potential toxicity of contaminants and any residual product from the manufacturing process. So you see, the task is hard. Perhaps this slide seems very basic for a lot of you. But I think it's good to mention the GLP. And there are a lot of new players in the vaccine field which need to take into account this requirement. GLP is a quality system which is applicable to non-clinical safety. And therefore, non-clinical safety studies must be conducted under GLP. And this includes in vivo toxicity studies--I mean animal studies, either single or repeated dose toxicity studies--but also, all the in vitro tests, all the in vitro toxicity studies performed on vaccines, such as genotoxicity tests for adjuvant, or any new in vitro tests. However, as is mentioned in the ICH Guideline S6, some part of non-clinical safety studies using very specialized test systems, such as immunological assays, may not be able to comply with GLP. And in this case, you can clearly mention in your product that one specific test will be performed outside GLP. So what are the prerequisites for animal toxicity studies to enter in phase I for vaccine candidates? The logic would say that we should start with acute toxicity study. However, it is not always strictly needed, because sometimes you can get this kind of information from your quality control test battery. Plus, you will get data from general safety tests. And therefore, in a lot of cases we start directly with the pivotal repeated dose study mimicking the human immunization schedule. And this repeated dose will be really a strong support to start the phase I. It's usually performed in one species, but we will try to add in this repeated dose study a lot of parameters in order to collect the maximum information. We will add immunological investigations, and I will explain that later. And we will also do some local investigation. We will do the histopathological evaluation of the injection site, in order to get some information on local tolerance; and avoid perhaps to perform another study just for this kind of investigation. And then, on a case-by-case basis, as it was mentioned this morning, we will add some other parameters, such as safety pharmacology; viral shedding, if we are dealing with a live virus; or biodistribution evaluation, if we are dealing with a genetically modified organism. So let's speak a little bit about the protocol for this repeated dose study. As I mentioned before, the key rule is to try to mimic to be as close as possible to the human immunization schedule. So we will prefer a sequential treatment, versus a daily administration. For example, I used to give the product every two weeks. This is true for the vaccine. We will see that for the evaluation of a new adjuvant. We may come back to the classical rule of daily administration for a new drug entity. We will also try to maximize the exposure. And I usually have one additional injection, as recommended by the FDA, compared to the human design. If possible, we use the human route of administration. And in the study, I am used to include two necropsy time points: one early, one or two days after the last vaccine administration; and another later time point, two to three weeks after the last administration. Regarding the number of animals, I mean, we use the rules used for all types of drugs. Usually, we use ten rodents per sex and per time point. And if we are using monkeys, it's two to three monkeys per sex and per time point. About the selection of the relevant species, I think it's really the essential question. And I hope that we will have a lot of discussion about this point. Brian will present some slides and will discuss the various options and the logic in the selection of the animal species. But I would like here to present some advantages and disadvantages of some species. It is true that the rat is the preferred species for toxicologists. I mean, we have a lot of background data in this species. It's a middle-sized animal, which allows in a lot of cases to inject one human dose per animal, and also to collect a sufficient amount of blood sample. However, we don't have always some immunogenicity data, and we have to acquire this immunogenicity data. And immunologists prefer in fact the mouse. But as far as toxicity, the mouse is, unfortunately, a very small species with limitation regarding samples. Concerning the rabbit, I know that it is an historical species, and I know that the FDA likes this species. However, it's a very delicate species, and we have few background data in general toxicology for this species. Regarding the monkey, it's probably the gold standard. We have a lot of information regarding the monkey immune system. And there is a close homology with the human immune system. However, it's an expensive species. Another question which will probably be discussed today is the number of dose levels: Should we use one dose level or two dose levels? If we use one dose level today we are generally using the highest possible dose level, using one human dose per animal or, if we can achieve that, we use the maximum feasible volume in the selected species. However, there is some tendency to think about another dose level. And in this case, the lowest dose level could correspond to the pharmacological dose. I mean a dose triggering an immune response in the selected species. What about the parameters? And Elizabeth listed some of them. I think for the parameters we should follow the guidelines already existing for classical pharmaceuticals. In my study I'm used to including body weights, with a weekly evaluation; clinical signs daily; body temperature, particularly in non-human primates, and on several occasions after the first and subsequent treatments; ophthalmological examination; cardiovascular examination, mainly in non-human primates, in order to include safety pharmacology parameters during the repeated dose study; hematology and serum clinical chemistry data; necropsy time points. Again, we can discuss today about the usefulness to add this type of analysis during the course of the study. Is it sufficient to have data at the end of the study, or do we have to add satellite animals to obtain this type of clinical pathology information? And then necropsy: We do a full necropsy, with microscopic examination of the tissues and organs. We measure organ weights, and we do a histopathological examination for quite a large list of tissues. Immunogenicity: I mentioned also that we have to add this type of evaluation for vaccine toxicology studies for several reasons. The first reason is to confirm and to justify the selected species. We need a species which reacts to the vaccine. It's also a good way to confirm the vaccine administration, as we are not doing pharmacokinetics or toxicokinetics in this study. And also, it's an additional proof of concept of the vaccine in an animal model. There are two types of responses which can be evaluated: the humoral response, by ELISA or ELISPOT assay, or by other types of tests, such as neutralization tests. Usually, the humoral response is evaluated in serum samples. But sometimes you can also use nasal or vaginal lavage for the evaluation of the mucosal response. The cell mediated immune response is more complex to evaluate, particularly because we don't have always the right reagent in animals. And it's also very difficult to collect the cells, and to protect the cells, and to do this assay very rapidly. The methodology: ELISPOT assay, or the intracellular cytokine detection. What about the timing for this pivotal toxicology study supporting the future of phase I clinical trials? Usually, to design the study protocol we need to know some information about the clinical protocol. So it's sort of a "Catch-22" situation, because we cannot start a toxicology study without information about the next step. But I think it's clear that the toxicology design will be based on the number of administrations in humans, on the targeted population, etcetera. So we need to obtain this information from your colleagues from the clinical department. As mentioned this morning, there is a possibility to discuss the protocol with regulatory agencies; and perhaps particularly for non-conventional studies. If we send all study protocols to the FDA, I think they will have a huge amount of work. Then we can initiate the in-life phase in the study as soon as the product is available. I usually prefer to work on a clinical batch, but it is not strictly necessary. We can work on a dedicated batch, either GMP or GMP-like. And then, additional tests can be needed. But they can be performed prior to the phase I, or later on during the development of the vaccine before the licensing or before phase II/III trials. And just to illustrate all these recommendations, I have put here one example of a monkey study, and I have selected one of the most complex designs. You have here a prime boost strategy, with priming with GMO, in fact, with a Canarypox vector expressing the vaccine antigen. And then we really mimicked the human design by repeating this prime boost strategy. And we have the boost with the antigen alone. And as you can see, we have reproduced here the human treatment scale with various groups in the study and various frequencies of administration. As I mentioned before, we have added quite a lot of parameters, classical toxicology parameters, such as ophthalmology, cardiovascular examination, clinical pathology. But we have also added humoral and cell-mediated immunogenicity on-point. As we are dealing with a live virus, we have added a viral shedding evaluation, to measure the shedding of the virus in the environment. And we have also added at the end of the study biodistribution evaluation by quantitative PCR, as we are dealing with a TMO administration. And this study was sufficient to support a phase I trial in humans. Sometimes we have to add some specific investigations to this classical toxicology study. And it is really on a case-by-case basis. I have tried to present here some examples. But I think it will be very difficult in a guideline to list all potential tests which can be required to assess the safety of a vaccine. I have here just mentioned what we did for meningococcal vaccine. This was a vaccine using the transferrin-binding protein. And one of our questions was: Would antibody against neisserial transferrin receptor cross-react with human transferring receptor? And therefore, in order to document this question, we did first a literature search, in order to see if there are autoimmune disorders associated with meningococcal disease. And we didn't find any data about this potential link. Then we worked on computer in order to do sequence analysis, and we compared the sequence analysis, the sequence alignment, between neisserial and human receptor. And we didn't find any sequence homology or similarity. And then, in addition to this literature search and then to the computer evaluation, we did some in vitro experiments in order to study the potential cross reactivity of antibodies from the vaccine on human tissues by fluorocytometry. And we didn't observe any cross reactivity. I have listed here all the examples of specific investigations which can be required either before phase I or later on during the development of the vaccine. It could be the evaluation of antibody dependent enhancement assay for Dengue vaccines. It could be the evaluation of disease exacerbation model for RSV vaccine; viscerotropism evaluation for yellow fever vaccine. And I think the list is long. It really depends; it's really on a case-by-case basis. But what to do if there is no evident relevant animal model? This could be case, for example, for Dengue vaccine or small pox vaccine. First, in my opinion--but I will be very happy to share a discussion with all the people in the room--in my opinion, it's very difficult to claim that there is no animal model at all. Then, perhaps to reduce the number of animals used, but still to do something, you can combine an immunogenicity study with general toxicity; with viral shedding, if we are dealing with a live virus. We can also try to reduce the number of animal uses; perhaps to use only one species and to reduce this number to, for example, two-plus-two per group for monkeys; and to mimic exactly the human design, perhaps one single administration, and therefore a very short study. As was also mentioned before, not all the studies are required before phase I. Some of them can be performed later on. And during the day, we will speak about the evaluation of the risk of autoimmune diseases. And Paul Henri Lambert and Mike Luster will in their presentation present this risk and the methods available. Also, later during the development of the vaccine, the developmental toxicity studies can be performed. And this will be the subject of tomorrow. And also, for clinically modified organisms we may have to perform biodistribution evaluations. This is true for GMO, and also for naked DNA vaccine. Brian I think will present a case study on this issue. This evaluation is intended to detect exposure of non-targeted organs, particularly germ-lined tissues, to exogenous DNA. And the method used is a quantitative PCR on tissue fragments from dedicated studies or dedicated organs. And if tissue or organs are positive by quantitative PCR, an integration evaluation is needed for the remaining positive samples. Last but not least--and I think that this subject will be also exposed by Natalie Garcon--what are the requirements if we develop a new adjuvant or a new excipient? And my position is in this case to first define the toxicology profile of the adjuvant or the excipient alone, by doing toxicology studies as we do for new chemical entities. I mean acute toxicity studies in rodents by IV route or IP route; repeated dose with daily administration in two species; pharmacokinetic evaluation; genotoxicity tests; and any other specific tests related to the structure or to the mechanism of action of the adjuvant or the excipient. And then, when we have the toxicology profile of this product, we have also to combine this product with a vaccine and to test the combination in a repeated-dose toxicity study. We have also to verify the pharmacokinetic of the adjuvant when it is combined in the vaccine formulation; and also, to perform on this combination the other studies requested for the adjuvanted vaccine. Okay. So now I would like to conclude on this presentation by saying that a few decades ago vaccines were considered as safe, ipso facto. I think it's clear for all of us that today vaccine safety is thoroughly evaluated as well as all pharmaceuticals. And I'd like also to put some more emphasis on vaccine safety evaluation. I like this sentence recently published in "Nature," saying that, "Predictions based solely upon epidemiological projections without solid scientific bases are often misleading." I think it's clear that there are a lot of arguments justifying science-based non-clinical safety evaluation for vaccines. However, there are some remaining gaps between the existing tools, the existing toxicology methods, and an ideal, fully relevant preclinical safety evaluation. And to finish my talk, I have tried to list here some potential corrective actions. First, by doing a toxicology study we will learn, and learning comes from performing these types of studies. Second, potential corrective action: Perhaps we should encourage academic groups to make research in this field. I am thinking about the users of juvenile animals and the very interesting research performed in Geneva by Dr. Carol Sieglitz [ph] in this field. Also, we need also perhaps to encourage to boost collaborative research and validation programs. I am thinking about the ILSI initiatives already done for classical drugs. Perhaps similar initiatives need also to be started for vaccine safety evaluation. Thank you very much. [Applause.] DR. VERDIER: Do we have burning questions before the coffee break? Natalie? [Question Inaudible.] DR. VERDIER: I think we need a sufficient number of data showing the quality of the preparation of the product. When you will make your safety study, you need to archive with your raw data the information showing that you have prepared your product according to good manufacturing procedures. [Question Inaudible.] DR. VERDIER: For me, GLP is really limited to the safety end points. So you cannot use the word "GLP" for the manufacturing of the product. But it's true that you have to follow the GLP recommendations when you will manufacture your dedicated toxicology batch. [Question Inaudible.] DR. VERDIER: I am used to adding in my monkey studies ECG evaluation, plus obviously histopathological evaluation of the herd. In a recent study, we observed some histopathological changes in the herd. And that's why it could be interesting to see if these histopathological changes have consequences on the ECG. We measure ECG and blood pressure. PARTICIPANT [In Audience]: We find measuring these [inaudible] monkey studies [inaudible] studies to be very unreliable; very difficult to interpret that data, and often misleading [inaudible]. Changes are difficult [inaudible]. DR. VERDIER: No, I cannot really comment. I mean, I used to do that, and it's performed for classical drugs. So I don't have any argument to say that vaccines are really different. I mean, do you think that a dedicated safety pharmacology study will be better for that? PARTICIPANT [In Audience]: I don't really want to go down that path. I mean, if you have concerns [inaudible] I might consider that. DR. VERDIER: I can tell you that in this case we did a dedicated safety pharmacology study. PARTICIPANT [In Audience]: So if you use that, if you use that approach, what's the point of including [inaudible] study? I mean, the only thing you can see is a very negative effect [inaudible], which I suppose is something. But it would have to be pretty dramatic to be able to see it [inaudible]. DR. VERDIER: You are perhaps right. I'm used to doing it only for primate studies. Jean Villain [ph]? [Question Inaudible.] PARTICIPANT [In Audience]: It sounds like you may have to repeat that question. Or maybe you can go to the microphone. Because I think people in the back may have trouble [inaudible]. PARTICIPANT [In Audience]: My question to Francois was what he means with the cardiovascular importance introduced in the toxicity study, especially in primates. And when I was involved at the RAVM in the vaccine studies on pharmacology and toxicology, we found out that there were important cardiovascular effects on the blood pressure of the classical pertussis vaccines. And I am wondering whether this is a more general feeling in the vaccines, or whether it has been studied even? DR. VERDIER: Well, I confirm that we do blood pressure and ECT. PARTICIPANT [In Audience]: Yes. You mentioned in talking about dose, dose appropriate to generated immune response in the specific animal that was being used. However, the position of the FDA is that the dose should be the human dose; not something less, not dose per kilogram, but the actual human dose. So is this a specific disagreement that you have with the FDA? DR. VERDIER: No. When I'm using only one dose level, I am using one human dose per animal, or the maximum physical volume. If, for example, I have in mind a mouse study, in the mouse you cannot always achieve this one human dose per animal. So in this case, you will give the maximum volume. I am not in favor of changing the formulation of the vaccine. Because one way would be to increase the antigen concentration in order to have the one human dose per mouse. But in this case, you change totally your vaccine formulation. So in rats, in rabbits, in primates, in a lot of these middle-sized species, you can for your highest dose--or perhaps for your unique dose, if you are just using one dose--achieve this one human dose per animal. My remark was in the case of several dose levels in the same study. In this case, yes, the second dose could be just a pharmacological dose level. DR. MIDTHUN: If I could say a word, Francois? I think the subject on dose is one that we plan to discuss in the roundtable discussion. And so you may want to defer that discussion until that time, at greater length. PARTICIPANT [In Audience]: Francois, I've got to--Can I ask you a very nice question? Since you are asking toxicological questions, why one species? DR. VERDIER: It's a very interesting question. I hope that we will discuss that today. PARTICIPANT [In Audience]: And he thanks me to ask him. DR. VERDIER: We say one relevant species, because we think that it's already difficult to select one relevant species. So the second species could be less relevant than the one you have selected. So you have really an argument to say that one species is more relevant than another one. In this case, why do a second species in a lower model, if you wish? But it's true that if we cannot differentiate the relevance between a monkey species and a rat species, then perhaps you will have to perform a second species; but later on, not to support your phase one. Perhaps to support your licensing. PARTICIPANT [In Audience]: In your table of species, the pluses and minuses of using different species, you didn't include guinea pigs. Any reason for that? DR. VERDIER: It's a good question. I will speak about guinea pigs this afternoon for hypersensitivity reaction. I think for general toxicological studies, it's quite difficult to use guinea pigs, because we don't have--It's a little bit like the rabbit: We don't have a lot of background data in guinea pigs. It's a very delicate species. However, with some vaccines--I have in mind, for example, CMV vaccines. There are some publications about CMV vaccine and guinea pigs. So then it's really on a case-by-case. However, I should tell you that I've never used guinea pigs for a general toxicology study, until now. PARTICIPANT [In Audience]: Have you given a look at the liver; as there are old studies on BCG and the effects on Hexobarbital duration, effects on Hexobarbital duration, sleeping time. Is there anyone that has included this type of evaluation in their vaccines? DR. VERDIER: I didn't get all the words. You mentioned the liver? PARTICIPANT [In Audience]: The liver, the liver metabolism, and Hexobarbital sleeping time is affected by the [inaudible] vaccine, and maybe also by pertussis vaccine. DR. VERDIER: No, we didn't do this type of specific assays. We have, obviously, the liver as part of the organs for the histopathological examination. We have also some liver enzymes as part of the clinical chemistry parameters. But we don't do any functional assays on the liver. We focus on hypertoxicity for some vaccines. I have in mind a yellow fever vaccine and [inaudible] vaccine using the yellow fever virus. In this case, we do some investigation on the liver, but it's mainly in vitro assays, rather than additional parameters in animal studies. I think if there are no more questions, it's time for coffee break. Thank you very much. [Applause.] [Morning Recess.] DR.Sutkowski: I think we'd like to get going again. I do have a couple of announcements. I just wanted to point out, I neglected to say at the beginning of the meeting that this whole meeting is being transcribed. So by that I mean, obviously, not videotaped, but audiocassette recorded, and then it will be transcribed. And we hope to make the transcription available, along with the speakers' slides, available to you all possibly on some website that either the SOT or CBER would set up, to make the transcription summary or the actual transcription available and the speakers' slides available to you all. I also would like to remind you that this is not a regulatory meeting. It is instead a scientific workshop. And we sincerely hope you have come here to help us work on refining our approach to non-clinical safety assessment of new vaccines and adjuvants. We would like to get your views and, if possible, try to reach some sort of consensus on the most appropriate and most feasible methodologies that can be used to determine the potential adverse effects of new vaccines and adjuvants, appropriate animal models for these evaluations, and the utility of these data for the design and conduct of clinical trials. And once again, regarding the transcription, we would like to ask, if you wouldn't mind, to please state the question and then possibly the moderator will repeat the question. And if you would like to give your name and affiliation, that might be helpful, as well. Also, in terms of how we envision these sessions to run, each session will begin with the chairperson giving a brief presentation to introduce and provide a general overview of the specific topic. And then following that, the chairperson may choose to present a case study, to go over aspects of a particular product for the purposes of providing an example of a product type to be discussed from a fundamental point of view. Then the topic will be opened up for an interactive discussion. And during that time, we invite you to discuss the topic. We would like to try to keep the discussion focused on the fundamental questions; rather than discussing the particular nuances of individual product categories. You may either present your questions over the microphone or, if you prefer, you could turn in index cards to some of the SOT staff members. Let's see, what else? We do know that there is going to be overlap in the issues discussed in the various sessions; but where possible, we ask that you try to stick to the topic at hand. And if the moderator thinks that the question may be more appropriate for another session, we may ask you to defer the question. For example, in general, I would like to suggest that we not discuss the various questions with respect to adjuvants until we get to the adjuvant session. And if there are no other questions in terms of clarification, then let's begin with topic one. I'd like to call on Sally Hargus, the regulatory toxicologist within our Office of Vaccines Research and Review in CBER at the FDA, who is going to be the moderator for session one. ROUNDTABLE DISCUSSION: SCIENTIFIC AND TECHNICAL CHALLENGES IN PRECLINICAL SAFETY TESTING OF VACCINES DR. HARGUS: Hello, everyone. I would like to welcome today Dr. Brian Ledwith. Brian comes to us from Biologic Safety Assessment at Merck, where he is the director. Brian has a B.S. in chemistry from William and Mary. He got his Ph.D. in biochemistry from the Medical College of Virginia, and an MBA recently from the Wharton School, U. of Penn. He also did post-doctoral work at Merck in safety assessment, under Matt Bradley [ph] and Warren Nichols [ph]. Brian, thank you for agreeing to make this presentation on relevance of animal studies for non-clinical safety evaluation of vaccines. THE RELEVANCE OF ANIMAL STUDIES FOR NON-CLINICAL SAFETY EVALUATION OF VACCINES PRESENTER: BRIAN LEDWITH, DIRECTOR, BIOLOGIC SAFETY ASSESSMENT, MERCK DR. LEDWITH: Thanks, Sally. I'd first like to just give a very general overview into some of the concepts in choosing an animal model for carrying out these studies, touching on some of the points that Francois touched on earlier; but then use our studies of our adenovirus-vectored HIV vaccines as a case study, not to provide you so much particular data to those studies, but really use it as a tool to demonstrate our criteria for selecting animal models and other study design factors in developing these preclinical safety studies. So of course, it's first very important when deciding on the animal models to really determine: What are the objectives in our preclinical safety studies? And of course, the fundamental objective is to provide compelling evidence to support the introduction of a vaccine into human subjects. And it's important that we realize that in the phase I population it's generally healthy individuals, and so our animal models are generally healthy animal model systems. And we have an understanding that many rare toxicities, idiosyncratic effects, or potential effects on certain sub-populations in the human population are generally often only addressable in humans. So by and large, animal toxicity studies focus on generally healthy animal models. It's also important to realize in the design of these studies that we're trying to maximize the benefit-to-risk ratio of developing a vaccine; which means we want to minimize the risk by rigorous safety studies, but we also want to proceed with timely development of important vaccines that can affect human health. Of course, one of the fundamental things we need to do is determine a safe dose for the phase I trials. And this is basically a no-effect level for toxicity, with an acceptable safety margin for humans. But here it's important to realize, by "no-effect level" we're talking about significant toxicity, and not the desired immune response. And in designing the studies, it's important to realize, again, we want very broad measures, because most toxicities are unpredictable. But in certain cases there will be a key theoretical concern for a certain vaccine type. And in those cases, it may be warned to include specific assays that may be more sensitive, or at least additional approaches for addressing those key theoretical concerns. So for considerations for choosing an animal model, as we've discussed already, a major focus is on the relevance of the animal model with respect to immunogenicity; that it demonstrates the expected immune response that you're looking for in people. This could be a humoral response, or a cell-mediated response, or both. And when choosing the animal model, it may be important to understand that for not all species--particularly rabbits for cell immune responses--reagents may be limited. We want to have a sensitive animal model. Hopefully, that way we've demonstrated that it does respond to immune-mediated effects, or has been shown to be susceptible to the intrinsic toxicity of the test article. It's also important to realize that the intrinsic toxicity can be separated from immune-mediated toxicity. A classical example, of course, is pertussis toxin vaccines, where we use an activated pertussis toxin to remove the intrinsic toxicity. And this is tested in certain release tests. And hopefully, the only effects you'll see are the immune-mediated effects. But two of the most important criteria from a safety assessment perspective are using models where you have experience, where you have large historical control data bases, so that you can interpret sporadic findings to determine whether they're just sporadic changes in a control incidence, or really a treatment-related effect. And it's also important to be consistent, because we want to develop correlations with our preclinical animal models with respect to the clinical safety of those products when they reach the clinic. Some additional considerations that have already been raised are whether it's important to do single or multiple species; whether we should use inbred or outbred strains when we're using rodents. At Merck we tend to use outbred strains because we feel it's preferable to have that diversity and heterogeneity in the test animals. So for mice we generally use CD-1 mice; for rats, Spraig [ph] Valley rats. And of course, we are concerned about species- or strain-specific sensitivity. And that's when possibly a second model may be of value. As Francois alluded to, there are certain advantages between using large animal models versus the small animals. We tend to be able to use a larger number of animals per group when we're dealing with rodents. However, the disadvantage there is that we may need separate groups of animals for separate end points; whereas in a large animal model, all end points could be carried out in the same animal. Again, the issue of dose comes when there is a desire to inject a full human dose. Then you're almost exclusively restricted to a larger animal model. But the disadvantage there, because of limitations in the amount of test article, etcetera, is that you generally have limited body weight margins, safety margins based on body weight. And you can actually have much more exaggerated body weight margins in the rodents, which I'll show you in the case study. And of course, additional models may be needed on a case-by-case basis, as Francois alluded to. An example of this would be a cancer vaccine contained in the self-antigen, where you may want to test a self-antigen in animals, meaning using the animal homologue of that test animal antigen. [Tape Change.] DR. HARGUS: Any other comments? [No Response.] DR. HARGUS: Okay. MR. BARKER [In Audience]: Lee Barker [ph], Sequella [ph] Foundation. The second speaker said it's hard to make a case for there not being a relevant species. And I'm wondering how important, in considering whether a species is relevant, the panel would consider natural disease produced by the microbe of interest simulating human disease. Because that can make it relatively difficult to find a relevant animal. And another very specific question that I think was touched on towards the end by the first speaker, Dr. Sutkowski, and that is a great many vaccines are given to either newborns or very close to newborn humans. So I'm interested in hearing some discussion about whether the relevant age is newborn or suckling animals. And I'm not sure how commonly that's practiced, but I'd like to hear some discussion of that. Thank you. DR. HARGUS: Thank you. Who would like to take that? Francois? DR. VERDIER: I think you touched here on a very important question: Do we have to use juvenile animals for a pediatric vaccine? You know probably that there are new guidelines for pediatric drugs. I think today we need to get more information about the immune system of juvenile animal models. We are not yet ready to use these juvenile animals in toxicology. And that's why I was mentioning at the end of my presentation that fundamental research should be performed on juvenile animals. The problem also is the feasibility of this model. I mean, if you give the drug by oral route, that's fine. Or by, perhaps, IV; that's fine. You can use juvenile animals. But if you give the vaccine by IM, I don't see how we can treat juvenile animals. To give you also another example, for a vaccine intended for elderly people we used aged mice. So we altered mice at four weeks, and then we kept these mice for nearly six months in order to start a study on six-month-aged mice. MR.         : Yes, I just want to comment on what Francois brought up about. There is a draft guidance on juvenile animal studies to support clinical trials with drugs. That guidance I think is probably going to be published pretty soon. But in there there is no statement that you should routinely do juvenile animal studies. You do it on a case-by-case basis. And I don't know, that's probably what's going to wind up with vaccines. DR. HARGUS: Yes, another comment? DR. VERDIER: Yes, I would like to have another comment on this topic, since we are very much involved in this type of work using very young animals. And one important point if we deal with vaccine is to consider the immune status and the development of the immune system soon after birth. It's clear that a neonatal mouse has nothing to do with a neonatal infant. On the other hand, what we tend to see now is that a one-week-old mouse is much closer to the human infant, in terms of development of the structure of the lymphoid organs, the appearance of follicular and [inaudible] cells, possibly of developing an immune response. In fact, you find quite a lot of the deficiencies which can be seen in the newborn are seen also in a one-week-old mouse. To what extent this can be used for toxicology and to assess the potential risk that we have there, I think that there is a whole bunch of work to be done there. And we know that for some adjuvants it's probably important to look at young animals as well, because we see different types of reactions. But the knowledge is still quite limited. MR.         : I was actually going to touch on this in my talk, too, about small molecule programs. And I think we're still approaching the subject of whether it's feasible, whether it's relevant, whether the model is relevant, whether it's feasible to do the dosing. With small molecule programs you certainly can make the argument about differences in metabolism versus--you know, young versus old animals. Here we talk about differences in immune response, young versus old animals. It's not clear to me that we are ready to jump off that and try to do those studies now with vaccines. How we get to a point where we might be able to address that question is open for debate. DR. GRUBER: Well, I just wanted to add a point. I really think we would agree that we are not there yet, asking for toxicity studies to include juvenile animal models to assess the safety of a vaccine that is indicated for an infant population. And not to say that there shouldn't be research encouraged in that field, but the approach that is usually taken, besides doing a toxicity study, is to then do your first clinical trial in adults, and stepping down to toddlers, and then to infants, to gather more on the safety data in older humans before you then go and do your studies in infants. And I think that's the approach that is currently taken, and is something that is probably going to be employed for a while before we are at the point that we can entertain the idea of using juvenile animal models for assessing the safety of a product for the purpose of moving into a phase I clinical trial. DR. HARGUS: Shall we move on to the next question? Go ahead, Stu. MR. SHAPIRO [In Audience]: Yes, Stuart Shapiro [ph], from the Division of AIDS at National Institute of Allergy and Infectious Diseases. I'd just like the panel to address the differences in safety testing between therapeutic and preventive vaccines. I notice that the guidance that's being developed is specifically for preventive vaccines. However, we increasingly see people--and it's not the large drug companies, but it's mostly academic investigators--who have this idea that if they come in first with a therapeutic vaccine, first of all, it won't be reviewed by the same people--which I keep telling them this is not true. It's a vaccine for an infectious disease; it's going to get to the Office of Vaccines. But secondly, they have the feeling that if they start off testing their vaccine as a therapeutic vaccine, the requirements for tox testing will not be as great; and then once they've had it in ten or 20 humans, they can turn around and say, "Oh, look, it's safe, it's got a safety profile." But we know, those of us who have some experience with it, that they don't get the level of data that you get from doing a thorough preclinical tox study where you can necropsy the animals, sacrifice them at the end of the study and do thorough necropsies. And just the level of information you get is not the same. So I would hope that you could first shed some light on the FDA's thinking about this; and secondly, that when you're writing this guidance document, you take that into account, that this issue really needs to be addressed. DR. HARGUS: Okay, I'll give this a shot. I can tell you that we don't get that many therapeutic vaccine INDs. By and large, we get--Right. Right. I mean, this is an emerging area. And typically, the approach that we've taken is to be consistent in requesting a definitive preclinical GLP safety tox study for everything now; not across the board, but for a novel preventive vaccine, for a novel therapeutic vaccine. And we would expect that the sponsors would provide us with an adequately designed preclinical safety study prior to going into phase I, which would typically be in an adult healthy population. And then after phase I, you would go into maybe a very small group of your target population. And then you would take it from there. But at this point, we are consistently requesting the up-front definitive safety study in a preclinical model. MR. SHAPIRO [In Audience]: Then your guidance document should probably be for preventive/therapeutic vaccines. DR. HARGUS: Well, I think we'd have to clarify what kind of therapeutic vaccine. And we'll certainly take that under advisement. Thanks. Oh, Liz has something to say. DR. SUTKOWSKI: I think perhaps if Mercedes has anything to add, that she should feel free to do so. But our program, in terms of how it's evolving, we have consulted all along with Dr. Dave Green's group in the Office of Therapeutic Vaccines, and we are striving to put this program in writing for vaccines. But we've consulted with them all along, and we are trying to be consistent. And if Mercedes has something to add--? DR. SERABIAN [In Audience]: Yes. This is Mercedes Serabian. I was with the Office of Therapeutics. I'm now with the Office of Cell Tissue and Gene Therapy. When you say "therapeutic vaccines," I guess the first thing I think of is for cancer, because that's generally the indications that come in to us; not for infectious diseases, if you will. Again, you have to consider risk-benefit. Many of these--obviously, cancer, a life-threatening disease. It depends on what's required preclinically. At times there may not be a relevant animal model. It may be just in vitro studies that are done. It depends on your product and your-- [Question Inaudible.] DR. SERABIAN [In Audience]: For AIDS? Well, I guess it would--You tell me, Karen. It would go to your group? DR. MIDTHUN: Yes. DR. SERABIAN [In Audience]: Yes. So I mean, I honestly can't respond, except through OTR experience. But the ultimate call would be with the OVRR group. You're correct on that, yes. DR. HARGUS: Thank you, everyone. Let's move on to the next-- PARTICIPANT [In Audience]: Actually, I--Is it true? Do you think that you would deal with a therapeutic vaccine for an infectious agent differently than as a preventative? DR. MIDTHUN: Yes. This is Karen Midthun. I guess, just to say a few words, yes, it's correct. Office of Vaccines would have vaccines against infectious diseases that are for therapeutic indications, also. And our approach would be to view those vaccines with the same safety considerations as we would view vaccines for the prevention. So that I think the same issues and considerations would go into that, also. DR. HARGUS: Okay. MS. CHRISTIAN [In Audience]: Mildred Christian, Argus [ph] Research. Well, we'll be spending a day tomorrow looking at reproductive considerations. I think that the speakers this morning also lead into the conditions in which one must consider that many of these vaccines will be given to potentially pregnant women, and also to pregnant women, and to pediatric populations, as ultimate populations that are to be treated. And I'd like to follow the rationale for species selection a little bit. Because last year at a similar meeting on selection of animal species and testing for developmental immunotoxicity, we decided that it was not appropriate to use Balb/c mice most of the immunotox models that are standard will use for developmental tox studies, based on there not being sufficient historical data. What I noticed was that developmental tox, when requested--And it will be requested more frequently, because pregnant women will be the test population. When tested, there is a tendency to go to the rabbit, which is fine. But in that case, one is attempting to potentiate the immune response to the maximum amount, either by giving boosters, or testing pre-pregnancy. And I wondered if the speakers would address the rationale and say whether they conducted sub-chronic or other tests to show when the maximum amount of immune response occurred in an alternative species; since usually the primate is not practical for these tests, and a different mouse strain would be what they had as information in their companies for developmental tox background. MR.         : I can say something. So in contrast to the other representatives from industry, we are doing most of our tox testing in rabbits. To specifically address your question--And again, I was going to talk about this in my talk. But you know, we don't do traditional pushing to MTD and those kinds of things; or even daily dosing, which may be an issue in terms of designing a developmental tox study. But we focused on the rabbit. There are issues with historical database, but we do get a complete tox package in rabbits. So we're going down that path. DR. LEDWITH: In our approach we haven't actually carried out a DART study yet, but we are planning to in the next year or so. And our approach would be to use our validated model that's in-house, which would be the rat, and demonstrate the relevant immune response in the rat. So rather than trying to adapt the DART testing to an immunogenicity model, validate the DART testing model that we have established in-house by demonstrating the relevant immune response. DR. HARGUS: Okay, thank you. Let's move on. Sir? MR. GREENBLAT [In Audience]: Jay Greenblat [ph], National Cancer Institute. The case study presented, you had no virus-vectored vaccine. And a significant portion of the human population already has an immune response that had no virus. I was wondering if you thought it would be beneficial to include a group of animals that were pre-immunized who had no virus, or a similar vector? DR. LEDWITH: We haven't done that in our toxicity studies because, all the evidence we had, that would only diminish the response to the adenovirus vector from a toxicological perspective. As I showed you in my talk, almost all of the findings related to toxicity were most pronounced after the first dose; and many not even observed after subsequent immunizations when the animals have neutralizing antibodies against the vaccine. The issue raised is certainly an issue in the human efficacy trials. And that's certainly under consideration there. MR. GREENBLAT [In Audience]: Thank you. DR. VERDIER: To your question regarding several positive animals, I can give you another example. For RSV vaccine, we did studies with sero-positive and sero-negative animals, in order to study both cases. MR.         : To a certain extent, we've addressed the question, and it may cross over to another question, about a rechallenge experiment, whether we need to put in a rechallenge dose. In reality, the repeat dose tox study, you might even think of that as a--you know, a challenge in the face of an ongoing immune response, or a preexisting immune response. And perhaps even a more rigorous test would be a rest period and then a rechallenge; which is one of the questions I think we're going to try to talk about, too, about the relevance of that. But that in my mind sort of gets at that same question: whether the toxicity, in the face of an ongoing immune response, is different, or a preexisting immune response is different, than in the absence. MR.         : Well, that makes me wonder about a particular issue which Brian had mentioned. And that is, he had conducted tox testing both in animals that you knew were undergoing an active immune response, and hence you had seen changes in lymphoid organs that you might expect: larger lymph nodes, larger spleens--Which, you know, you sort of cast over, saying, "Well, they were minimal." But it's something you'd probably want to see, I think, in an active vaccine. DR. LEDWITH: Right. MR.         : I don't think it's something to be worried about. And then, the issue then is, if you're doing standard toxicity studies where you're going into a higher dose of the vaccine to look for unwanted toxicities, would you be also measuring whether there is an immune response occurring at that particular time? Because I think it would be unlikely, or it's a possibility that if you're in high dose you're going to be inducing tolerance and you won't be seeing that in your response. So how do you distinguish between the changes you see, whether they're toxic or normal changes, that occur from a vaccine in an animal that's undergoing an immune response, versus those that are not undergoing an immune response? Because they're going to be quite different. I mean, do you monitor that? DR. LEDWITH: We address it in the sense that the doses that we've used in our animal models are similar to--or are identical to the doses that were carried out in the immunogenicity experiments. So we know that under the dosing regimen we're using for our vaccine, for example, we're not seeing tolerance; that we've already characterized the extent of the immune response, whether we're seeing a boost or whether we're seeing basically a steady duration of the immune response. So those particular concerns really haven't come up in our studies. MR.         : But classically, then, what you're measuring is the toxicity of immune response. And if you're looking at a classical toxicity study where, for whatever reason, you go to a higher dose--most sensitive individuals you're protecting, or whatever--you're not really addressing that specific question, right? DR. LEDWITH: Yes, and this gets back to a little bit of what Francois was saying. We're limited by vaccine formulations, compared to small molecules, in being able to push the dose towards what I think you're suggesting, maximum tolerated dose types of things, where we will see overt toxicities. And basically, what we can do is push to the highest dose that we can in these animal models, and then evaluate the toxicities with respect to whether they're expected, with respect to the immune response, or whether there are really organ-specific toxicities which really are not related to the desired effect of the vaccine. DR. HARGUS: Okay. I think we'll take one more question, and then we're going to have to move on to the next speaker, in the interest of time. And for those of you who didn't get a chance to ask your question, please write it down on one of the cards and submit it to us. Or, if you want to wait until the next session--I mean the next discussion period after Garvin, then you can ask your question then. Go ahead, sir. MR. SNOW [In Audience]: Yes. My name is Bill Snow [ph], AIDS Vaccine Advocacy Coalition. And I'd like to go back to the basic question of this panel, which has to do with the animal models. The case study that was presented, presented three animal models: the mouse and the rabbit and the monkey. And each was serving a different purpose. The question that I have is in the introductory tox, that was not required. I'm wondering under what circumstances--For example, if you had monkey immunology data from a preclinical lot that made the company confident to move forward, when could you cut back from those three? And under what circumstances in an exploratory vaccine program would you be able to get some human data before going to all of the expense and time of doing these tests and the manufacturing at the GMP level? DR. LEDWITH: I'll take a first stab at that, because it was my talk that prompted the question. First of all, our choice of using those three different animal models was basically voluntary. It was not required upon us. That was basically our study design, which we did discuss with CBER prior to the initiation of the studies. And we chose to do two animal models for complete toxicity basically because, as many of you are probably familiar, there was great controversy about the use of adenovirus vectors in a gene therapy trial at that time, and significant safety concerns, some of which we felt could only really be evaluated in monkeys. So we wanted to have both our more standard, small-molecule mouse toxicity study, combined with a second species, Rhesus monkeys, to adjust really for those particular safety concerns for adenoviral vectors. We don't do both species routinely for all vaccines. And with respect to having an additional model for local tolerance, again, that was more of a voluntary, in-house procedure, because we've used rabbits for so long. Now, rabbits was not the best model for us for a full toxicity study. It's not validated at Merck for tox studies. But we have used it for intramuscular studies. So we just chose to use that for local tolerance. But we also evaluate local tolerance in the tox studies. So it's quite possible in a single study to address the local tolerance issues as well as the systemic tox issues. DR. HARGUS: Ken, do you want to go ahead? MR.         : Yes, just one comment I'd like to make, since you brought it up. One of the things--And I'll just make the comment, and this can be discussed later. I am wondering a little bit about whether the issue of systemic inflammatory response really was dealt with to the depth that I would have expected it to in this preclinical model. You know, you look at the overall data that was presented, and there's a little glimmer of things going on. And when you're talking about a small number of animals, you want to explore that a little bit more in depth. That's all I want to say about that. MS         : I just had a more general comment. I thought--And perhaps I'm challenging the panel members and the audience here a little bit. But the session was about the relevance of animal studies and animal models. And I think what we heard today is that animal studies are critical and necessary to get a feeling about the toxicity profile of a vaccine. But what I haven't really heard this morning is a discussion about what the relevant animal model really is; and what the relevant immune response really is; and if we really should stick with the note that was made earlier to say we need to work with animal models for which we have a large amount of historical background and experience, so that we can interpret some sporadic adverse events that we otherwise in a non-traditional species would not be able to interpret. But my question is if we perhaps have to compromise here a little bit and say, okay, we may not have a species that is validated by all means in that we have a large amount of historical data and background data, but that we know it's valid and that the immune response that is induced is somehow relevant to what we are really getting at. And perhaps we need to discuss it a little bit more; if not today, then we can do it tomorrow. Because the issues are somewhat similar, I think, and we can perhaps revisit this tomorrow. Because I think that there is a case to be made perhaps to really take a stab at looking at perhaps not the well established animal data in order to arrive at a more relevant animal model. And that's a comment I had to make. DR. HARGUS: Okay. Thank you. At this point, I'd like to introduce the second speaker for this session, Dr. Garvin Warner. Dr. Warner received his Ph.D. in microbiology and immunology in 1986 from the Albany Medical College, and did a post-doc and was a research assistant professor in David Scott's lab at the University of Rochester Cancer Center. In 1991, he joined drug safety evaluation in Bristol-Myers Squibb in Syracuse, and expanded the immunotoxicology and exploratory toxicology group there as part of the department of biologics evaluation, and was responsible for the early drug safety and development programs for a number of immunomodulatory, oncology, and therapeutic vaccine programs. In 1997, he moved to Genetics Institute, Andover, and was responsible for a number of development programs for therapeutic protein. After Genetics Institute was fully incorporated into Wyeth Pharmaceuticals, then American Home Products, he was responsible for development programs in immunology and hemophilia. He is currently the director of exploratory drug safety, Wyeth Research, Andover, Massachusetts, and is responsible within drug safety and metabolism for the biopharmaceutical, hemophilia, and vaccine programs. Okay. And Dr. Warner, thank you very much for agreeing to make this presentation on the applicability of traditional drug toxicity study designs for safety evaluation of vaccines. APPLICABILITY OF TRADITIONAL DRUG TOXICITY STUDY DESIGNS FOR SAFETY EVALUATION OF VACCINES PRESENTER: GARVIN WARNER, DIRECTOR, EXPLORATORY DRUG SAFETY, WYETH RESEARCH DR. WARNER: I have to apologize right off the bat because I may confuse people, in the sense that I tend to play devil's advocate. And you may say, "I thought he said this, and now he's saying that." So bear with me on that. I'll try to generate some discussion here. Words to live by. I often run into this problem when I'm dealing with--because I do cross over into the discovery groups--of why we're doing toxicity testing when we know that this is okay. This isn't going to be a problem. Why do you push that dose so high? You know, we don't need to do that; that's ridiculous. But the reality is, we do toxicity testing to look for unexpected effects, not for expected effects. We use scientific judgment and try to think about what we might see, but the reality is we're looking for unexpected effects. So I just really want to give you a little bit of view of drug safety evaluation from a traditional small-molecule company perspective and touch on: What's the point in a traditional tox study, or tox program; factors that influence the traditional toxicity program; selection of species--some of these topics are going to overlap with what we talked about last time--general flow in our tox safety programs; and then sort of move on to whether vaccines fit the drug toxicity testing paradigm. In the concept of maximizing exposure to the test article, does one size--I think we've already answered that question. Does one kind of study fit every kind of program? I think that's clearly "No." The kinds of study designs: I will present a sort of straw dog study design at the end, really not from a viral--you know, from a sub-unit perspective for a vaccine program. Issues related to the immunogen versus the adjuvant--we'll talk about adjuvant later--versus immunomodulator. I throw that in. Now people are starting to throw other things in besides an adjuvant; perhaps direct TH1/TH2 responses. And then I've also put up here--and it's interesting, it's been brought up several times--the test article used in IND enabling studies. What do we need to have for that, in order to do our GLP/IND enabling tox studies? So in a small-molecule program we establish an MTD. We push the dose to the point where we see toxicity. Indeed, some would argue that you haven't done a toxicity study unless you have toxicity. Otherwise, you've just shown that it's safe at that given dose. So with most small-molecule programs, we can push the dose to an MTD. And we usually do it in two species--We always do it in at least two species. And primarily, we're trying to identify target organs of toxicity, to guide clinical research into what to look for in their clinical studies. We establish a safe starting dose based on the no-toxic-effect level, or the no-effect level in the tox study. And the focus really is on exposure. We maximize exposure. We measure exposure. We do pharmacokinetics. We can talk about exposure relative to the area under the curve of our tox dose versus our pharmacologically active dose; which of course is a little bit of a problem in vaccines. We can change dose levels. You know, we're non-restricted usually in the formulation; although that can happen at times. We can increase the number of doses relative to the clinical study. We can change the schedule. We can change the route to maximize; go IV, if we want to look at a maximum systemic exposure. And the studies are staged with increasingly longer durations of treatment to support the clinical program, and ultimately to support registration. There are some factors that influence the study design, and perhaps the timing of the studies as we do them. But the reality is it's pretty--I don't want to say it's really straightforward, but it's reasonably straightforward about what we have to do for all of our small-molecule programs. But there are issues, you know. If we're treating a terminal disease, oncology, that program may look different than treating asthma, for instance. You know, whether it's non-life-threatening disease; and whether there are other existing therapies. Age of population: We touched on it already. We'll talk about women of child-bearing potential tomorrow in developmental toxicity, but I also put up here juvenile and pediatric studies. Now, with small-molecule programs, of course, we would do a juvenile study generally, prior to going into infants, or to less than--whatever, 16 years old. With vaccines, the history has been that we don't do those studies. And again, as I said before, I'm not sure that they're really relevant in the context of a vaccine program, or whether we have enough information to know that they're relevant. And of course, the duration of treatment: If we're talking about a drug for an acute indication, a single-dose study, that program will look different than one for chronic lifetime administration. So we generally pick the most sensitive species in terms of any toxicity noted. I'm really giving my view here. And in terms of rodents, we prefer rats; but occasionally mouse. And certainly, it's often needed for carcinogenicity studies. Our non-rodent, we prefer dog; but sometimes we use non-human primates, or sometimes we do both. And again--I'll jump down here--often the selection has to do with the relative metabolism in the two primary tox species, and the exposure. So generally, we'll either use the most sensitive species and/or the species that gives us the greatest exposure. So again, everything is exposure based. I'm going to briefly touch on proteins. You know, historically, everybody says, well, proteins are dealt with differently than small molecules. And certainly, they have different issues. But again, and I bring up some of the issues that cross over into the vaccine area, right? So we need to demonstrate pharmacologic activity in the species selected. Often, particularly with monoclone antibodies, an MTD can't be established. Okay? So in contrast to the small-molecule program, we can't reach an MTD. And the question always comes up: When do you stop dosing? When do you stop escalating your dose? Often we end up with, as has been mentioned here before, dosing based on maximum feasible dose, based on the formulation that can be provided to us. And the toxicities seen here are often associated with the pharmacology; like the vaccine programs where we're often worried about the response and not so much--well, at least historically, anyway--the intrinsic toxicity of the molecules themselves. But we're able to measure exposure here. And generally, the greater the exposure, the more rigorous the test. And the question is: When do you stop dosing? When do you stop pushing that dose? Immunogenicity: I'll bring it up. Here we've talked about tolerance already. Early on, immunogenicity was a big concern, "Oh, I can't dose. If I get antibodies in my animal model, I can't dose any more." Well, you know, that turns out to be maybe not the case. You can either dose through the immune response; maybe you induce some tolerance; maybe--I don't like that word; I'm an old "tolerance" guy. But "clonal exhaustion," whatever you want to cal it. But there's an example where, pushing the dose, clearly you can get over issues of immunogenicity. It's relevant to vaccines in the sense that pushing the dose in a vaccine may be the exact opposite way of where you really want to go. So the general flow here, we do exploratory, non-GLP studies, often single-dose MTDs, with toxicokinetics, range finders, repeat dose. Sometimes these are GLP. And exploratory metabolism. In phase zero, our IND enabling GLP tox studies, including the full battery of gene tox, generally a 28-day tox study with TK. Some safety pharmacology. We've touched on that. We do dedicated safety pharmacology, cardiovascular safety pharmacology studies. And the reproductive toxicity studies to support women of child-bearing potential. And of course, the timing of these is depending on the clinical population. And that basically is phases I through III. We're just keeping up with the clinic, trying to extend the duration of our studies to cover the duration in the clinical studies; and then start adding in things, depending on clinical plan: juvenile studies, chronic studies, reproductive toxicity studies, and ultimately carcinogenicity studies. So do vaccines fit the drug toxicity testing paradigm? Does the concept of maximizing exposure to the test article, does it apply to vaccines? Now, we've got to separate out adjuvants. And are we really trying to do two things with our studies? Are we trying to not only assess the intrinsic toxicity, but we're also trying to assess the--I don't know, the pharmacodynamic toxicity, the toxicity of the pharmacodynamic response to the response [sic]. So the question to me is: What is the test article? Is it the adjuvant? I would argue that it's both; it's the adjuvant, and it's the combination. We look for it in the intended immunologic consequence. That is either anti-immunogen antibodies; the cell-mediated response, as several of the speakers have already talked about. The question of: Will maximizing the exposure to the immunogen actually hinder the intended immunologic response? So Francois talked about using a dose--and we do this, also--a dose that is intended to maximize the intended response, and a dose that we do several-fold over. And often, it is based on the formulation, the maximum feasible dose. And of course, I should say in these animals that we're talking about mgs-per-K--or mgs-per-meter-squared, or however you want to gauge it--we typically dose on a per-dose basis, using the equivalent human dose if we can, and then some multiple of that. But built into that is a safety margin based on mg-per-K, as we've already mentioned, particularly in mice. Vaccines are a complex and diverse class of products. And immunogens--I mean, we've had sub-unit vaccines; purified; recombinant DNA derived; live attenuated viruses, which have their own issues; CD&A vaccines; vector vaccines; and chemically synthesized vaccines and adjuvants. So clearly, for this gamut of things it isn't a "one size fits all" kind of a toxicity study that we can use for everything. Dose selection? Again, we've already mentioned that a couple of times. I bring up the issue of a special tox formulation. That's come up several times in our discussions. We know what the clinical formulation is, but if we want to push the dose, should we come up with a special tox formulation where we've increased the concentration of the immunogen? Schedule: Maximize exposure again, like in a small-molecule program. Honestly, I would never think of going daily dosing in a vaccine study, even with an adjuvant. And we can talk about that later. And then the question is: Do we trade off--And again, we're doing two things with the study. We trade off an optimum mean response for an optimized exposure to the test article. Route has come up. Intended clinical route only? I would say, yes. Other routes of administration to maximize systemic exposure: At times, is that a relevant thing to do? I mentioned before, sometimes we'll go IV, even with the small-molecule program, to maximize systemic exposure. Is there a place in a vaccine program to do that, also? And then, the duration: Generally, as has been brought up several times--We call it the "N-plus-1" convention, the number of clinical doses, plus one. What also has come up is the rechallenge issue, whether we should build into studies a rest period and then a rechallenge. Because often these vaccines will be used perhaps more than one sequence of immunizations during a lifetime. Immunogen, versus adjuvant, versus immunomodulator: Do we need to assess individual toxicity in each one of these things? In general, I'll just give you a paradigm for our sub-unit vaccines. In our tox studies we include an adjuvant alone, the immunogen alone, and then combinations of the immunogen and the adjuvant. Usually, when we do the immunogen alone, it's a top dose, in the combination arm of the study. And if we're doing two dose levels of the adjuvant, it's at the top dose of the adjuvant. And we'll discuss the novel adjuvants and the adjuvant issues later. So finally, I just want to touch on this issue of test article used in the IND enabling studies. This is less of a problem for the small-molecule program people because by the time we get our stuff, they're talking about their first 50-kilogram batch and things like that. But there are questions regarding the contaminant profile. It's come up in a couple of other talks, too. So we're not only assessing the activity or the toxicity of the active ingredients, but also of the contaminant profile. So this needs to be representative of the clinical material. It should have a similar activity profile. It may not be GMP; it may not be fully GMP compliant. But what data should we have to say that it's representative of the clinical material? Especially given that often at the time that you run into tox studies, you don't have the clinical material yet. A similar biochemical, biophysical profile, is that sufficient? And of course, the question that always comes up in dealing with protein programs, too, is: When is a change a change? So scale, ten-fold scale, a new peak on size occlusion chromatography, those kinds of issues. And this is what was also touched on a little bit, too: Sometimes the clinic is used to decide which gets brought forward and how much preclinical work is necessary to do those human immunogenicity studies. I mean, I think HIV is a particular example of that, where we're bringing a lot of things forward, in hopes that our human studies will tell us what works best, or what is likely to work best. And the question is, in a life-threatening disease like that, is there some minimal amount of work that we can get into a phase I study? So here's my straw dog of a tox study. And maybe we could just leave this up here and, again, perhaps open the floor to questions. So I'm talking about several questions. So actually, up to here is a four-dose study. You know, typically, N-plus-1. We're going to do four doses. The question: Interval; how long between doses? I've heard two weeks mentioned. We've done four weeks, because that's what the most aggressive schedule in the clinical is going to be. What should that decision be based on? What should the decision on dose be based on? Early necropsy? How long after that last dose should we do the necropsy? Should we do two necropsies? Should we look at one--I would argue that perhaps conducting a necropsy at an optimum point, when the immune response is expected, is the appropriate place to do it. I certainly haven't listed all the clinical end points here, and there were other ones here. But I'm really lumping everything together because, in honesty, the tox studies that we do--which I've already mentioned, we often do do rabbits--it's a full tox panel that we would do for a small-molecule program. A full histo; we add in measuring immunologic parameters associated with the pharmacology. But it's a full tox path assessment. So I don't know, questions from the floor? Do you want to open up the questions again? Really a continuation from the last questions. DR. HARGUS: Let's thank Garvin. [Applause.] [Tape Change.] DR. HARGUS: --discuss the topic of animal models along with the topic of study design considerations. Let's open the floor to questions addressing those two broad issues. MR. VAN DER LAAN [In Audience]: I have first a question on the--Jan-Willem van der Laan. I am a member of the safety work part of the CPMP. With respect to the dose and the dose level and the number of dosages, I'm not sure--and maybe the immunologists on the panel can answer--whether the immune response is dose dependent. I expect at least a bit. And if Dr. Verdier is indicating that one dose is sufficient, how to be sure if you have to do yet the phase I, how to be sure what is the human dose. And I think that our questions which came up during this lecture of Dr. Garvin [sic]. DR. WARNER: I think that is a good point. And Francois mentioned trying to get the clinical plan before designing the tox studies, which is sort of related. But you're right, that top clinical dose is a guess, honestly. I mean, in the sense that we're talking animal models, and the reality is that we don't know that it's going to be relevant to the human dose, the needed human dose. So, you know, it's sort of a development question for the people in development. Because talking to the research people, they're saying, "Ah, this will work." And that may be true. But we do try to--I try to influence people to build in some safety margin there: the highest anticipated clinical dose, and then some multiple of that. So that when we get into the clinic, we have some room to move; and that the tox study supports dosing at higher levels than based on the mouse immunogenicity--or in our case, we do rabbit immunogenicity. PARTICIPANT [In Audience]: Garvin, does it matter whether or not you use body surface area or body weight? DR. WARNER: So, okay, if you use mgs-per-meter-squared, all you're doing is building in an extra factor. PARTICIPANT [In Audience]: Yes. DR. WARNER: If you want to do that, say you've got to, instead of using tenfold based on mgs-per-kg, just say use 30. The basis for mgs-per-meter-squared for small-molecules is based on metabolism and clearance. At least, that's the argument. We don't know here whether that's relevant or not. If you want to build in additional safety margin, just build it in based on mgs-per-kg. That's my opinion. DR. VERDIER: Jan-Willem, I agree with you that there is not a clear dose relationship for the immune response. And that's why perhaps one human dose is perhaps sufficient, because with high dose or low dose you will trigger the same humoral and cellular mediated response. There are some cases where you have to test two doses. If your vaccine is mixed with an adjuvant, one human dose per animal could be too high, and you may have to test lower doses in order to avoid a very severe inflammatory reaction due to your adjuvant composition. And also, I think I would like to emphasize the fact that I'm not really in favor of changing the vaccine formulation to test higher concentration, even if this is a way for new chemicals. DR. GRUBER: If I can comment on that, too, indeed, when you have a formulation, and especially when you have the antigen--and I am more talking about recombinant antigen--and you have a specific formulation, the interactions between each component defines your formulation. If you start going by mg-per-meter-square, or by weight, it doesn't mean anything any more in your formulation. You change completely the system, and you may see responses that are not relevant to what you would see for the human response. So from our perspective, it's better to stay with one human dose, which in a rat or a rabbit is already in vast excess of what you would have in a human. DR. WARNER: I agree. I'm not advocating it. I'll play devil's advocate, though: It depends. It depends on the adjuvant that you're using. It depends on whether you can get stability at a higher concentration. I was really thinking in my mind when I thought this through of maintaining a constant adjuvant level, and then changing the concentration of immunogen, and perhaps doing that in exploratory studies. So we could even define the immunogenicity profile, and see whether it's relevant. Again, I'm not advocating it. I'm just playing devil's advocate. MS. NOVIKI [In Audience]: Hi. I'm Deborah Noviki [ph], from Chyron [ph] Corporation. A lot of the comments that I was going to raise just got raised as the discussion was occurring. But I wanted to sort of just raise a point, and it was covered a little bit with the last comment. And exploratory studies are not really exploratory for Chyron, but the immunogenicity studies that actually justify the utilization of an adjuvant. I depend greatly on the preliminary pharmacology and immunogenicity studies that my colleagues are running. And that actually helps drive a great amount of the way I design a study to do the toxicology. I, too, use rabbits extensively, because primates and mice tend to be the species that our pharmacology and immunogenicity studies are done with. And what I've tried to do is work very closely with colleagues in immunogenicity studies and actually incorporate some safety end points into especially primate studies; not very much in mice at all. But I think that working closely with those groups, utilizing long-term studies that toxicologists would almost never run--I mean, we'll run primate studies for a year or more sometimes. And those are important opportunities to garner data, even if it's not done under GLP and even if it's done with research formulation. So I think that's a really important thing to think about. MR.         : I agree completely. And not just for vaccine programs, either; other programs, too. There's a lot of potential safety information that can be gotten out of well designed pharmacology or, in this case, immunogenicity studies. DR. HARGUS: Okay. Go ahead. MR. GILMER [In Audience]: Yes. My name is Ian Gilmer [ph], from the EPA. I was interested in the "N-plus-one" concept. Where I work we're interested also in cumulative and aggregate risk. Kids get anywhere from a dozen to 16 injections of different vaccines over a two-year kind of span. So I'm just wondering about the kind of cumulative effect of the different vaccines. And perhaps this is more relevant to the adjuvant, mainly aluminum hydroxide. But you know, there seems to be more of an exposure than what's being tested, if you look at what's going on in real life. MS.         : So if I understand your question, it's more in the light of pediatric schedule vaccination, considering all the vaccines they get. What you're suggesting is that maybe toxicity studies should be done in light of those vaccines, and you should somehow prime the animal with those vaccines before testing your new vaccine, or co-administer? MR. GILMER [In Audience]: Well, I'm interested in how the FDA--or