Preclinical development strategies for novel gene therapeutic products.

With over 220 investigational new drug applications currently active, gene therapy represents one of the fastest growing areas in biotherapeutic research. Initially conceived for replacing defective genes in diseases such as cystic fibrosis or inborn errors of metabolism with genes encoding the normal, or wild-type, gene product, gene therapy has expanded into other novel applications such as treatment of cancer or cardiovascular disease, where the risk:benefit profiles may be more acceptable in relation to the severity of the disease. Different types of vectors, including modified retroviruses, adenoviruses, adenovirus-associated viruses, and herpesviruses and plasmid DNA, are used to transfer foreign genetic material into patients' cells or tissues. Developing a toxicology program to determine the safety of these agents, therefore, requires a modified approach that encompasses the pharmacology and toxicity of both the gene product itself and the vector system used for delivery in the context of the application for the clinical trial. In general, the issues involved in designing and developing appropriate preclinical testing to determine the safety of these products are similar to those encountered for other recombinant molecules, including protein biotherapeutics. Limitations to some of the typical toxicology studies conducted for a traditional drug development program may exist for these agents, and nontraditional approaches may be required to demonstrate their safety. Many factors may affect the safety and clinical activity of these agents, including the route, frequency, and duration of exposure and the type of vector employed. Other safety considerations include quantitation of the duration and degree of expression of the vector in target and other tissues, the effects of gene expression on organ pathology and/or histology, evaluation of trafficking of gene-transduced cells or vector after injection, and interactions of the host immune system with the transduced cell population. Because of the unique concerns regarding each of these therapies, the Center for Biologics Evaluation and Research encourages sponsors to obtain toxicity data whenever possible while evaluating the pharmacologic activity of the vector in a species or animal model relevant to their clinical indication. Sponsors are encouraged to discuss preclinical study design and results with the Center during product development to facilitate early identification of safety concerns prior to entry of these novel agents into the clinical setting and to ensure an uninterrupted course of development while addressing issues required for licensure.

Safety assessment of biotechnology-derived pharmaceuticals: ICH and beyond.

Many scientific discussions, especially in the past 8 yr, have focused on definition of criteria for the optimal assessment of the preclinical toxicity of pharmaceuticals. With the current overlap of responsibility among centers within the Food and Drug Administration (FDA), uniformity of testing standards, when appropriate, would be desirable. These discussions have extended beyond the boundaries of the FDA and have culminated in the acceptance of formalized, internationally recognized guidances. The work of the International Committee on Harmonisation (ICH) and the initiatives developed by the FDA are important because they (a) represent a consensus scientific opinion, (b) promote consistency, (c) improve the quality of the studies performed, (d) assist the public sector in determining what may be generally acceptable to prepare product development plans, and (e) provide guidance for the sponsors in the design of preclinical toxicity studies. Disadvantages associated with such initiatives include (a) the establishment of a historical database that is difficult to relinquish, (b) the promotion of a check-the-box approach, i.e., a tendancy to perform only the minimum evaluation required by the guidelines, (c) the creation of a disincentive for industry to develop and validate new models, and (d) the creation of state-of-the-art guidances that may not allow for appropriate evaluation of novel therapies. The introduction of biotechnology-derived pharmaceuticals for clinical use has often required the application of unique approaches to assessing their safety in preclinical studies. There is much diversity among these products, which include the gene and cellular therapies, monoclonal antibodies, human-derived recombinant regulatory proteins, blood products, and vaccines. For many of the biological therapies, there will be unique product issues that may require specific modifications to protocol design and may raise additional safety concerns (e.g., immunogenicity). Guidances concerning the design of preclinical studies for such therapies are generally based on the clinical indication. Risk versus benefit decisions are made with an understanding of the nature of the patient population, the severity of disease, and the availability of alternative therapies. Key components of protocol design for preclinical studies addressing the risks of these agents include (a) a safe starting dose in humans, (b) identification of potential target organs, (c) identification of clinical parameters that should be monitored in humans, and (d) identification of at-risk populations. One of the distinct aspects of the safety evaluation of biotechnology-derived pharmaceuticals is the use of relevant and often nontraditional species and the use of animal models of disease in preclinical safety evaluation. Extensive contributions were made by the Center for Biologics Evaluation and Research to the ICH document on the safety of biotherapeutics, which is intended to provide worldwide guidance for a framework approach to the design and review of preclinical programs. Rational, scientifically sound study design and early identification of the potential safety concerns that may be anticipated in the clinical trial can result in preclinical data that facilitate use of these novel therapies for use in humans without duplication of effort or the unnecessary use of animals.

Biologically active luteinizing hormone is secreted in episodic pulsations that vary in relation to stage of the menstrual cycle.

To characterize the physiological pattern(s) of bioactive LH secretion in normal women, serial blood samples were withdrawn at frequent intervals in each of six women at three different stages of the menstrual cycle. Plasma LH concentrations were quantitated in each sample by both rat interstitial cell testosterone ( RICT ) bioassay and immunoassay (RIA). When the resulting RICT and RIA LH profiles were systematically compared, we found that mean (and integrated) plasma concentrations of bioactive LH were approximately 2-fold higher than immunoactive LH levels at all stages of the menstrual cycle. In addition, unequivocal prominent pulsations of bioactive LH could be demonstrated in these women throughout the normal menstrual cycle. For all stages of the menstrual cycle, bioactive to immunoactive LH ratios within LH pulses were significantly (P less than 0.01) increased over these ratios in the interpulse periods. The frequency of bioactive LH pulses increased dramatically from 0.44 +/- 0.24 (+/- SD) pulses/h in the early follicular phase to 1.21 +/- 0.07 pulses/h in the late follicular phase (P less than 0.003), and then declined to only 0.25 pulses/h in the luteal phase (P less than 0.001). Notably, significant discordance existed between bioactive and immunoactive LH pulses, with 30% of immunoactive and 14% of bioactive LH pulses occurring alone. In conclusion, using the RICT , we demonstrated that biologically active LH is secreted in discrete episodic pulsations in normal women. Estimates of the bioactive to immunoactive LH ratio indicate that these pulses of LH are preferentially enriched in biologically active compared to immunoactive hormone. The properties of bioactive LH pulses are under physiological control, since the amplitude and frequency of bioactive LH pulses vary significantly in relation to phases of the menstrual cycle. Since significant discordance exists between immunoactive and bioactive LH pulsations in normal women, we suggest that estimates of the circulating concentrations of biologically active LH (rather than immunoactive LH alone) are necessary to characterize fully physiological patterns of LH secretion during the menstrual cycle.

L-Proline transport by isolated renal tubules from newborn and adult rats.

Proline uptake and metabolism has been examined in newborn Sprague-Dawley rat kidney and compared to that in adult animals. [14C]-Proline uptake by renal tubule fragments from newborn rats occurs at the same initial rate as in adult tubules, but at physiologic concentrations achieves significantly higher intracellular levels after 15 min of incubation. Considerable metabolism of the proline taken up was observed in tissue of both ages. Analysis of acid soluble and insoluble tubule fractions from newborn and adults indicates similar degrees of proline incorporation into protein and oxidation to CO2 relative to the amount of radioactivity taken up. A major difference exists, however, with respect to the labeled components of the acid extract: adult tubules convert [14C]-proline to metabolites at a rate twice that of newborn. Analysis of concentration-dependent uptake data reveals two distinct entry systems for proline in both isolated newborn and adult tubules. No difference in the Km or Vmax was found between the young and mature tubules.

Transport of beta-hydroxy-beta-methyl-glutarate and beta-hydroxbutyrate by renal brushborder membrane vesicles.

The uptake of beta-hydroxy-beta-methyl-glutarate (HMG) and beta-hydroxy-butyrate (beta-HB) by renal brushborder membrane vesicles prepared from normal and starved rats was examined. HMG and beta-HB uptake show a Na+ gradient-induced overshoot, suggesting luminal cotransport of these organic acids. Kinetic analysis of HMG and beta-HB uptake revealed a single component carrier system and a diffusional component for each compound. Vesicles from starved rats exhibit the same transport characteristics as those from normal rats. The transport interactions of other organic acids with HMG were examined and revealed that citrate is a competitive inhibitor, which implies that the compounds share a common organic acid carrier.

The effects of diazene dicarboxylic acid bis-(N, N-dimethylamide) on glycine uptake by newborn renal cortex.

Glycine uptake by newborn rat renal cortical slices in the presence of 2 mM diamide remained unchanged from control value during incubation times up to 30 minutes. Longer exposure to diamide resulted in decreased net uptake. Kinetic analysis of this phenomenon indicated a noncompetitive inhibitory effect of diamide upon the low-affinity glycine uptake system, whereas glycine uptake on the high-affinity system was obliterated. A similar analysis of the diamide effects on adult tissue indicated a noncompetitive inhibition of both the low- and high-affinity glycine transport systems. Diamide did not cause any demonstrable change in glycine efflux from newborn cortical slices. Thus the decreased net uptake we observed in newborn slices in the presence of diamide could be explained solely on the basis of an effect on glycine entry. Simultaneous measurements of intracellular glutathione (GSH) levels in the newborn tissue showed a lack of any direct relationship between the transport effects of diamide and its oxidative effect on reduced glutathione.