James D. Green

Interspecies scaling of clearance and volume of distribution data for five therapeutic proteins

The clearance and volume of distribution of five human proteins (recombinant CD4, CD4 immuno-globulin G, growth hormone, tissue-plasminogen activator, and relaxin) in humans and laboratory animals were analyzed as a function of body weight using allometric scaling techniques. These proteins cover a 16-fold range of molecular weight (6 to 98 kD), are produced by recombinant or synthetic methods, and may be cleared by different mechanisms. The analyses revealed that the clearance and volume data for each protein were satisfactorily described by an allometric equation (Y = a Wb). The allometric exponent (b) for clearance (ml/min) ranged from 0.65 to 0.84, the allometric exponent for the initial volume of distribution (ml) ranged from 0.83 to 1.05, and the allometric exponent for the volume of distribution at steady state (ml) ranged from 0.84 to 1.02. Exponent values from 0.6 to 0.8 for clearance and 0.8 to 1.0 for volumes are frequently cited for small molecules and are expected based on empirical interspecies relationships. When the preclinical data were analyzed separately, the pre-clinical allometric relationships were usually predictive of the human results. These findings indicate that the clearance and volume of distribution of select biomacromolecules follow well-defined, size-related physiologic relationships, and preclinical pharmacokinetic studies provide reasonable estimates of human disposition. Employing this methodology during the early phases of drug development may provide a more rational basis for dose selection in the clinical environment.

Pathology of recombinant human transforming growth factor-beta 1 in rats and rabbits.

The systemic administration of high doses of rHuTGF-beta 1 to rats produced a spectrum of lesions in multiple target tissues, including liver, bone, kidney, heart, thymus, pancreas, stomach, cecum, at the injection vein, and in skeletal muscle at the site of anesthetic injection. The majority of these lesions can be attributed to known biological activities of TGF-beta 1. High-dose dermal application resulted in local effects at the wound sites without systemic toxicity.

Pharmacokinetics and Tissue Distribution of Recombinant Human Transforming Growth Factor Beta1 After Topical and Intravenous Administration in Male Rats

Recombinant human transforming growth factor beta (rhTGF-β1) enhances the healing process after topical application to various animal wound models. A detailed pharmacokinetic and tissue distribution study was performed to support the clinical development of rhTGF-β1 for wound healing indications. Rats received radioiodinated or unlabeled rhTGF-β1 as an intravenous (iv) bolus or as a topical formulation applied to a full thickness wound. Plasma concentrations of TGF-β1 were estimated from TCA-precipitable radioactivity or were measured by ELISA. Following iv administration, the initial half-life was rapid (<11 min), regardless of whether radi-olabeled or unlabeled rhTGF-β1 was used. The terminal half-life was long (163 min) when the test material was radioiodinated and administered as a trace dose and relatively short (≤61 min) when given at high doses and assayed by ELISA. Analysis of plasma radioactivity by SDS-PAGE revealed a time-dependent clearance of the 25-kDa parent molecule without a significant appearance of lower molecular weight radiolabeled metabolites. The majority of the radioactivity was associated with highly perfused organs, known iodide elimination pathways, and the thyroid at 1 and 8 hr after iv injection. After topical administration of a high dose (0.8 mg/kg), no immunoreactive TGF-β1 was detectable in plasma samples taken over a 48-hr period. However, trace amounts (≤0.05 ng/mL) of acid-precipitable radioactivity were detected in plasma after topical application of [125I]rhTGF-β1 (1 µg/kg, 126 µCi/kg). A significant portion (35%) of the [125I]rhTGF-β1 persisted intact (25 kDa) at the wound site 24 hr after application. In conclusion, rhTGF-β1 was rapidly cleared after iv bolus administration and distributed primarily to the liver, lungs, kidney, and spleen. Little systemic exposure was observed after applying a single topical dose of rhTGF-β1 to a wound, and the intact molecule persisted at the wound site.

Comparative Pathology of Recombinant Murine Interferon-γ in Mice and Recombinant Human Interferon-γ in Cynomolgus Monkeys

Publisher Summary This chapter discusses the data obtained from a series of toxicity studies with rHu (interferon) IFN- γ in a variety of species and presents a comparison of those findings with data from studies with recombinant murine IFN- γ (rMuIFN- γ ) in the mouse. It describes a complete series of subchronic toxicity studies and segment I and II reproductive studies in the rat, which revealed no evidence of toxicity at any of the doses tested. The results suggested that studies conducted in pharmacologically nonresponsive species may not be predictive of clinical toxicity. Human IFN- γ is active on nonhuman primate cells, though not at the same level as on human cells. Multidose studies in cynomolgus monkeys with rHuIFN- γ for 28 or 90 days are predictive of many dose-limiting clinical toxicities. Qualitative similarity is observed between toxicity studies employing rHuIFN- γ in the cynomolgus monkey and rMuIFN- γ in the mouse. The adverse effects seen in toxicity studies with cytokines and growth factors are often exaggerated pharmacological effects of the molecules and, therefore, can only be studied in a responsive species. In the situations in which a high degree of species specificity is encountered, studies employing a recombinant protein in a homologous species may provide a useful test system for preclinical safety assessment.

Issues with Biotechnology Products in Toxicologic Pathology

The emergence of the biotechnology industry and introduction of drugs derived from recombinant DNA technology has generated many new issues in approaches to preclinical safety evaluation and extrapolation of results to risk assessment in humans. Products or therapeutic approaches for consideration include hormones, growth factors, cytokines, monoclonal antibodies, vaccines, blood products, antisense, and gene therapy. In many instances the application of standard safety tests conventionally used for small molecules are of limited value or are inappropriate. Studies should be designed to answer specific scientific questions rather than simply to fulfill regulatory requirements. Special consideration must be given to study design and species selection in terms of biological activity and species specificity, implications of immunological responses in the animal studies, and effects of systemic administration of molecules at clinically relevant doses. A full understanding of the clinical relevance of toxicological and pathologic findings associated with administration of these molecules to laboratory animals requires definition of the pathogenic mechanism of lesion induction.

Utilization of homologous proteins to evaluate the safety of recombinant human proteins — case study: recombinant human interferon-gamma (rhIFN-γ)

Interferon-gamma is an immunomodulatory cytokine that has an extremely restricted host range of activities. RhIFN-gamma was one of the first species-specific recombinant proteins to be assessed in conventional safety models typically utilized for xenobiotics. Acute, subchronic and Segment I and II reproductive studies in rats revealed no evidence of toxicity at any of the doses tested; these results were not predictive of clinical toxicity, which is not unexpected since rodents are known to be pharmacologically nonresponsive to rhIFN-gamma. In contrast, 4- and 13-week multidose toxicity studies in cynomolgus monkeys with rhIFN- were predictive of many of the dose-limiting clinical toxicities. RhIFN- is active on non-human primate cells, though not at the same level as on human cells. In addition, qualitative similarities were observed between toxicity studies employing rhIFN-gamma in the cynomolgus monkey and recombinant murine interferon-gamma (rmuIFN-gamma) in the mouse. These results suggest that in situations where a high degree of species specificity is encountered, studies employing a recombinant protein in a homologous species may provide a useful test system for preclinical safety assessment. This information should be evaluated in conjunction with data from studies conducted with the human protein in pharmacologically responsive animal models when possible.

Preclinical Safety Assessment Considerations in Vaccine Development

No vaccine is totally safe and totally effective, and adverse reactions have been reported with all. However, adverse reactions to vaccines are generally mild, and severe events resulting in death or permanent damage are rare. Historically, for every approved vaccine, the benefits to the public of preventing the target disease far outweigh the risks and costs of vaccination (Duclos and Bentsi-Enchill, 1993). As the target disease disappears, however, rare adverse reactions to vaccines alter the risk: benefit ratio. Individuals become more concerned with the risk of adverse reactions to the vaccine than the risk of contracting the disease. Concern regarding adverse events from vaccination can have serious detrimental impact, as illustrated by events in the United Kingdom in the mid-1970s. Fear of the whole-cell pertussis vaccine was linked with vaccine complications ranging from fever to seizures, and the vaccine was even blamed for causing brain damage and death. This fear led to a 50% decline in immunizations. Several years later, pertussis cases began to increase significantly, which then led to a subsequent return to vaccination (Stuart-Harris, 1979). This event was, and still is, the subject of worldwide controversy.

Design of biological equivalence programs for therapeutic biotechnology products in clinical development: a perspective.

The determination of biological equivalence requires that studies are conducted to establish that two molecules, two formulations, or two dosing regimens, for example, are indistinguishable with respect to safety and efficacy profiles that have been previously established. The criteria that are used to establish biological equivalence will depend on the nature of the change (e.g., molecular, process, formulation), the stage of the development program, the duration of treatment, and the intended clinical indications. Key components of an equivalence program include chemical characterization, in vitro and in vivo bioactivity against reference material, pharmacokinetics, and safety. Special considerations for patient populations, endogenous concentrations, environmental factors, immunogenicity, assay methodology, biochemical identity, pharmacodynamic equivalence, and statistical methodology are discussed. In addition, the role of preclinical in vivo assessments is addressed. Specific case studies provide insight into the varied nature of approaches that are currently employed.

The Role of Pharmacokinetics and Pharmacodynamics in the Development of Therapeutic Proteins

Since the degree of preclinical work conducted to address safety issues in animals is usually rate limiting to the initiation of Phase I clinical studies, the preclinical program should be designed to allow rapid entry into the clinic without compromising safety. The establishment of acceptable therapeutic ratios relies on the relationship of doses utilized in toxicology studies and their projected relationship to the clinical dosing regimen. Experience with multiple therapeutic proteins (biomacromolecules) in our laboratory indicates that the pharmacokinetic behavior of many proteins is predictable across species. In selected cases, this information permits extrapolation of preclinical safety and efficacy data to the clinical setting when doses are related on the basis of pharmacokinetic equivalence rather than on a body weight (mg/kg) or body surface area (mg/m2) basis. With a better understanding of this cross species relationship, the confidence in the safety of a therapeutic agent in initial clinical studies is increased. Based on our experience, an approach is presented that maximizes the relevance of the preclinical information gained, minimizes the scope of the initial toxicology studies, and, therefore, minimizes time to initiation of Phase I. This strategy may be useful for selected classes of compounds. Also, several case studies are presented to illustrate how pharmacokinetics was used to bridge the gap between discovery research, preclinical studies, and clinical trials during the development of two therapeutic proteins (rCD4 and rCD4-IgG) for the treatment of Acquired Immunodeficiency Syndrome (AIDS).