Immune modulator studies in primates: the utility of flow cytometry and immunohistochemistry in the identification and characterization of immunotoxicity.

Exposure to natural environmental products, biopharmaceuticals, or investigational adjuvants has the potential to negatively impact the immune system, resulting in either up- or downregulation of immune function (immunomodulation). Many current protocols for primate toxicologic testing call for the evaluation of changes in immune cell number (peripheral blood or tissue), alterations in the weights of immune system organs (lymph nodes, spleen, thymus), and/or increases in the overall incidence of infections or neoplasms; these data are relied upon to suggest altered immune function. However, these are informative only when clear differences in frequency and/or severity of effects can be distinguished across control and dosed groups. In the absence of such distinct morphologic or clinical pathologic changes, the identification of potential immunomodulatory effects can present a much greater challenge. Additional evaluations may be needed to detect altered immune system integrity; these are based on in vivo assessments in primates of cellular or humoral responsiveness. Immunomodulatory effects can be characterized by in vitro or in vivo immune function tests: these tests require prestudy planning to integrate assessments into ongoing toxicology programs. These methods also involve specialized training and equipment, particularly if the intent is to evaluate parameters in a GLP laboratory setting. In primate toxicology, the added costs required to perform a complete functional analysis of the immune system can be substantial, but may be warranted depending on the clinical development plans. Two analytical methods that are easily incorporated into the standard toxicology profile in primates are flow cytometry and immunohistochemistry. Flow cytometry (FC) is used to assess changes in the relative distribution of immune cell marker expression, and where marker expression is known to fluctuate with the state of cell activation, can also provide information on functional attributes of immune cells. Immunohistochemistry (IHC) provides a means to evaluate similar characteristics of immune cells within tissue sections. Used together, FC and IHC can aid in the identification of changes in immune system that may not be apparent by traditional testing procedures (such as H&E staining), thus aiding in the characterization of immune system alterations. This presentation focused on the utility of flow cytometry and immunohistochemistry in a standard primate toxicology evaluation, with representative examples showing the benefits of these technologies in the diagnosis of potential immunomodulatory effects.

Overview status of preclinical safety assessment for immunomodulatory biopharmaceuticals.

Scientists from academia, industry, FDA, European and Japanese regulatory groups met to discuss key considerations that are central to the safe and expeditious development of novel biologic agents that are thought to act by modulation of the host immune system. In the presentations and case studies, particular attention was given to the current clinical experience with immunosuppressant agents. Many new biologic agents (such as humanized monoclonal antibodies) have been developed to interact in a highly specific manner with their target. However, their pharmacologic properties may be more complex than originally appreciated, impacting on clinical trial designs. The goal of preclinical safety assessment should be to provide some assurance that patients will be protected from any unacceptable risks by defining "safe" and "active" doses. For immunomodulatory molecules, particular attention is paid to defining potential for increased risks of lymphoproliferative disorders, opportunistic infections, and immune impairment. To address these issues, a wide variety of preclinical studies, mainly in non-human primates, have been performed for the purpose of assessing the potential risk of drug-induced, human immunotoxicity. Case studies presented at this symposium showed the feasibility of assessing humoral and cell-mediated aspects of the immune system, using antigen and neoantigen challenges, immunohistochemical, and flow cytometric (FACS) methods. In some cases, homologous forms of the biologic agent and "humanized" transgenic models have been used to assess potential clinical risks. These data have been useful in providing some assurance that severe adverse effects would not be induced in patients. Despite these limitations, it is important that industry sponsors provide information to regulatory authorities, the clinical investigator, and patients that provides the best feasible basis for risk assessment, safe clinical trial design, informed consent, and eventually, appropriate labeling. It is recognized that existing preclinical models often have significant limitations. Consequently, the sponsor's and regulatory authority's experienced judgement has determined whether or not the purported benefits of the novel therapeutic agent are balanced by the potential short- and long-term risks. In this field of development, preclinical models often need to reflect recent technology innovations; therefore, these models are not always "validated" in a conventional sense. Experience to date suggests that improved methods and approaches are needed as these agents are developed for use in lower or moderate risk patient populations. Consequently, there is an increased need for an industry/regulatory partnership in order to achieve progress in these risk assessment areas.

Regulatory decision strategy for entry of a novel biological therapeutic with a clinically unmonitorable toxicity into clinical trials: pre-IND meetings and a case example.

The following material was derived from a synthesis of case histories taken from investigational new drug (IND) applications and drug sponsors' experiences, utilizing fictionalized data to avoid any resemblance to any proprietary information; any such resemblance is accidental. These examples are used as an instructional scenario to illustrate appropriate handling of a difficult toxicology issue. In this scenario, a drug caused a toxicity in animals that was detected only by histopathologic analysis; if it were to develop in patients, no conventional clinical methods could be identified to monitor for it. It is not unusual for a firm to cancel clinical development plans for a lead drug candidate that causes such a toxicity, especially if such a drug is intended for use as a chronic therapeutic in a population of patients with a chronic disease. This case synthesis was inspired by a Food and Drug Administration (FDA) agreement to allow such a product to proceed into clinical trials after substantive pre-IND discussions and agreement on well-considered toxicology program designs. The scientists most closely involved in the strategy development included the sponsor's toxicologist, veterinary toxicologic pathologist, and pharmacokineticist, as well as the FDA's reviewing pharmacologist. The basis of this decision was thorough toxicity characterization (1-month studies in 2 species); correlating toxicities with a particular cumulative area under the curve (AUC) in both species; identification of the most sensitive species (the species that showed the lower AUC correlating with toxicity); allometric assessment of clearance of the drug in 3 nonhuman species; construction of a model of human kinetics (based on extrapolation from animal kinetics); and finally, estimation of clinical safety factors (ratios of the human estimated cumulative AUC at the proposed clinical doses, over the animal cumulative AUC that correlated with the no adverse effect levels). Industry and FDA scientists negotiated a joint assessment of risk and benefit in patients, resulting in the FDA permitting such a compound to enter into clinical trials for a serious autoimmune disease. Such constructive, early communication starts with the pre-IND meeting, and the conduct and planning for this meeting can be very important in establishing smooth scientific and regulatory groundwork for the future of a drug under IND investigation.

Elevated lead levels in a patient with sickle cell disease and inappropriate secretion of antidiuretic hormone.

A five-year-old girl with known sickle cell disease presented with severe hyponatremia and findings compatible with syndrome of inappropriate secretion of antidiuretic hormone (SIADH). She was found to have lead levels in the Class III category. By exclusion, we postulated that the SIADH was in some way related to the high lead levels, since this was the only abnormality the patient exhibited. The toxic lead levels and the elevated vasopressin levels rapidly responded to dimercaprol and calcium EDTA chelation therapy.

Prognostic variables in osteosarcoma: a multi-institutional study.

This is a report of a multi-institutional study of all patients with osteosarcoma who were seen at 13 comprehensive cancer centers from July 1, 1977, to December 31, 1982. Follow-up extended to 9 years; a minimum of 3 years was obtained for greater than 90% of the patients. All patients with osteosarcoma were considered, but only those with tissue confirmation who had had at least part of their first course of treatment at one of the 13 institutions were included. There were 543 patients. In a search for prognostic indicators, 38 patient characteristics, three treatment categories, and an institutional variable were studied. A combination of nine of these constituted the best indicator of survival. They were morphology (two parts), site of primary cancer (two parts), spread of tumor, grade and size of tumor, duration of symptoms, weight loss of greater than 4.5 kg (10 lb), swelling at primary site, and lytic appearance. Unexpectedly, treatment was not one of the indicators of survival. A prognostic score was developed in which the coefficients were obtained from the Cox regression (step-down) method. Each patient had a score (S) and an observed survival time that together provided the expected risk of death for that patient. Although this was not a randomized study, treatments were compared before and after adjusting for characteristics identified as prognostic. Three treatments differed little: surgery alone, surgery plus chemotherapy and/or radiotherapy, and chemotherapy and/or radiotherapy followed in 1-4 months by surgery. Patients with amputations and those with resections had similar death rates, but the observed progression rates differed widely. However, when the rates were adjusted for prognostic characteristics, the difference disappeared. Complete surgery (if osteosarcoma existed within surgical margins) was no better than incomplete surgery (if osteosarcoma existed beyond surgical margins) with respect to death but, as would be expected, complete surgery was much better with respect to disease progression.

Familial cancer in an oncology clinic.

Knowledge of cancer genetics provides the physician with a powerful tool for the recognition of patients who might profit from highly targeted cancer surveillance/management programs. Family history was evaluated by registered nurses on 565 consecutively ascertained patients with verified cancer from Creighton's Oncology Clinic. This initial assessment yielded 199 (35.5%) families with two more family members with cancer (all sites) within an informative nuclear component, which constituted parents, grandparents, aunts/uncles, siblings, and children. One or more of the operational criteria for cancer familiality, namely vertical transmission of cancer, bilaterality, and/or multiple primaries, early age of onset, and three or more site specific cancers, were found on physician review in 171 (30.5%) of the families. This group was referred for comprehensive cancer genetic evaluation consisting of pedigree extension and tumor verification through all second degree, and when possible, third degree relatives. It was determined that approximately 4% of the total clinic population demonstrated findings compatible with hereditary cancer syndromes. Its universal extension in clinical practice is advocated because of the potential yield from meticulous surveillance for cancer of highly targeted organs in such high-risk kindreds, as well as the economy and general case of obtaining detailed family history by registered nurses. The physician is able, therefore, to devote his primary effort toward pedigree analysis and syndrome identification.