Toxicology & Biotechnology at the Crossroads

Current issues in safety evaluation

By Steve Barkyoumb, DVM, Ph.D.; Chris Springall, M.D.; Friedhelm Vogel, Ph.D. and Richard Crowley Covance, Inc.

As a new generation of biotechnology-derived compounds enters the development pathway, there is a wide array of scientific and regulatory issues that are being addressed concerning the safety and efficacy of these compounds. The development and testing of these molecular entities poses challenges not found with traditional therapeutic compounds and requires a growing range of expertise and tools. Certainly the route of administration is seeing enormous interest due to the potential for the use of the inhalation route that seems to be particularly effective with protein-derived drugs. Another trend is the increasing importance of the nonhuman primate model in the evaluation of therapies for a wide range of maladies such as diabetes, osteoporosis, and neurodegenerative geriatric diseases. Responding to these issues creates challenges for regulators as they attempt to implement their public responsibility while responding to ever-evolving technologies and adhering to the goals of the FDA’s Critical Path Initiative. Each of these issues plays a key role in the presence and success of biotechnology derived compounds in the drug pipeline.

Drug Delivery for Next-Generation Therapeutics

Drug delivery has rapidly evolved from an academic discipline interested in technology innovation to a discipline focused on providing innovative therapeutic solutions. According to Dr. Josef Bossart, this evolution promises to deliver important benefits for patients, health providers and the biopharmaceutical industry. “An understanding of this promise can be found by looking at a product’s performance on parameters of efficacy, safety, convenience and price,” he remarked. In addition, the potential of a drug delivery technology can be estimated by its ability to enable, enhance, expand and transform the performance and exclusivity of an active ingredient.

The key objectives of any dosage form and delivery system include enabling a pharmaceutical to be used as a therapeutic as well as improving and enhancing its performance, and competitiveness. Drug delivery can also expand and transform the use of an approved therapeutic to provide for new applications. The commercial value of drug delivery is significantly impacted by the degree to which it can secure exclusivity. While a drug delivery technology can often be protected by patents, it is almost impossible to exclude technologies that can provide a similar benefit. Dr. Bossart observed, “Efficacy unarguably is the most important of the parameters in defining the performance of a pharmaceutical product. Without it, you have a placebo, but with efficacy a drug can be forgiven for sins of tolerability, convenience, price and even safety.” Where efficacy is a given, the most important considerations are safety and tolerability, which by themselves  can powerfully impact efficacy by enhancing compliance.

Improved convenience is often viewed as the greatest benefit associated with drug delivery. While improving the convenience of a product can be an important differentiator, it creates no leverage if it compromises safety or efficacy. The development of inhaled insulin is proving to be a tour de force for drug delivery that touches all of the key benefits of drug delivery including efficacy, which is positively impacted by the greater compliance associated with an inhaled rather than injected dosage form. The success of inhaled insulin will provide confirmation of the need for—and opportunity of—drug delivery  in providing next generation therapeutic solutions.

The commercial success of the iPod may offer a model for future drug delivery products. Rather than being the product of new, cutting-edge technologies, the iPod is the built with off-the-shelf components. Rather than focusing on new technology the iPod focuses on new applications and solutions. According to Dr. Bossart, the question becomes: Can Drug Delivery change the focus from new technology to applying the existing technologies to provide for new therapeutic options?  “In the next generation of drug delivery products the focus will need to go beyond enhancing the performance of pharmaceutical actives to expanding and transforming their applications. To be successful, companies exploiting drug delivery will need to take a therapeutic—rather than technological—focus. Technology will serve rather than lead,” he commented.

Developing Inhaled Biological Therapeutics

Large molecules are playing a significant role in the pulmonary drug delivery development market for the delivery to target lung disease, as well as in the use of the lung as a portal to the systemic circulation. Systemic delivery is aimed primarily at drugs that are currently delivered only by injection and that have permeability issues across the intestinal epithelium. These therapies present unique challenges not necessarily predicted from experiences with small molecules. Dr. Theresa Sweeney of Nektar Therapeutics noted that, “Con-siderations such as dose, target site, and patient requirements are important for choosing an appropriate delivery system for any type of inhaled therapy. As a result, an understanding of the advantages and limitations of different delivery systems for biological molecules will help the design of an appropriate program to evaluate the pharmacology and safety of molecules targeted for pulmonary delivery.”

There are several factors that affect total and regional lung dose, including the aerosol characteristics, lung anatomy and physiology, as well as whether it is intended for topical or systemic delivery. Technical challenges for optimal delivery of high dose inhaled biotherapeutics focus on devices that promote efficient delivery as well as consider patient convenience and variability. New delivery devices and formulation technologies effectively address many previously encountered compliance concerns, such as the concentration of drug and feedback systems for controlling breathing patterns thereby delivering a more consistent and effective dose. These technologies can efficiently deliver higher doses and offer the possibility of more precisely targeting the deep lung. To deliver large doses via dry powder inhalers, formulators must reduce interparticle cohesive forces to achieve improved flow and dispersibility.

Although there is no requirement to include the clinical delivery device in the animal toxicology/safety evaluation, inhalation administration should be done with a system that delivers an appropriate dose and particle size range. Dr. Jeffrey Tepper of Aerovance remarked, “Considerations for safety studies using novel delivery systems and devices should also include evaluation of excipients and assuring that the delivery system does not alter protein/peptide activity or integrity.” For biotechnology products, it is  beneficial if the delivery system is very efficient so as to conserve the (often very costly) active ingredient and allow evaluation at the high doses that may be required because of limited toxicology associated with many biotechnology products and the use of primates, rather than small laboratory animals.

The ICH S6 guideline for biotechnology products specifies the use of a comparable product to the product that will be tested in the clinic. For many companies, the limiting factors are both the cost and availability of material. Many biotherapeutics are expensive to manufacture even in small quantities, but the requirement to deliver a high lung dose to see any toxicity is a real financial challenge for many small biotechnology companies. The cost for a GLP lot of 50-1000 grams that may be required can be $0.5-$2M.This problem is further exacerbated by changes in the GMP process and incomplete formulation development after initial safety studies have been performed. Furthermore, the lack of cross reactivity with traditional laboratory animal species, as well as immunogenicity, can result in the use of larger species in particular the nonhuman primate. In addition to the standard issues for inhaled products such as the method of administration, particle size and determination of dose, the use of primates in safety pharmacology and toxicity studies does not always work well due to the small number of animals and lack of species-specific protocols.

To handle theses complex issues, the selection of a CRO with extensive expertise in working with nonhuman primates and with inhalation delivery experience is critical. The testing regimen will require safety pharmacology studies by inhalation as well as traditional toxicity studies.

When designing inhalation studies, the method of delivery (i.e., central plenum or individual exposure) is an important consideration in terms of the efficient use of test material as well as delivering consistent, predictable exposures. Either method can be used to administer the drug via the head or mouth/nose. If direct administration to the lung is necessary, individual administration can be done using an oral pharyngeal tube. Dr. Tepper noted each delivery device poses challenges: “The most critical of these is determining the actual dose delivered when taking into consideration the efficiency, losses due to inhaled volume fraction, and losses due to impaction in the oral pharynx. For EU studies, the amount presented (i.e., the amount available for breathing) is required, while the FDA requires the determination of the amount deposited in lung.” Clearance and accumulation must be evaluated by toxicokinetics to determine the systemic dose. The dose that penetrates the systemic system is partly determined by the physical chemistry properties of the molecule but is also influenced by the particle characteristics such as size, inertia, impaction, sedimentation and diffusion. Critical exposure measurements include the concentration and particle size.

Acute toxicity and respiratory safety pharmacology studies are done to determine tolerability, and alterations in ventilation. Additional methods to improve the understanding of respiratory toxicity include studies of respiratory mechanics, lung volumes and flows, as well as hypersensitivity and challenge models. Both the theoretical and practical aspects of delivering and measuring inhaled products have improved as has the efficiency of delivery. Measurement of bronchoalveolar lavage also can be used to improve the understanding of inflammatory changes associated with the clearance and toxicity of large doses of protein. Of particular concern is a better understanding of the antibody response to inhaled proteins. Whether inflammation associated with protein and antigen clearance is part of the normal homeostatic response of the lung or a prelude to more serious pulmonary toxicity requires further study.

Pharmacogenomic Biomarkers On Drug Labels

The scientific and regulatory paths by which pharmacogenomic data is included in drug labels focus on the evidence and benefit provided by knowledge of these data. Data on co-development of drugs and new tests requested for previously approved drugs may have different metrics and process maps by which they can be included in the label. According to Dr. Federico Goodsaid of CDER, “Several case studies showcase how pharmacogenomic data went from the lab to the label. There is a need to adopt a general process for updating labels with pharmacogenomic information.” This process includes developing the appropriate questions and capturing the relevant evidence to evaluate the quality of studies and the overall strength of evidence. Other factors must be considered in the relabeling decision to determine specific language for label. Some recent examples—such as mercaptopurines and irinotecan—support this proposal.

The million-dollar question remains: Will physicians use the pharmacogenomic data in drug labels? Says Dr. Goodsaid, “Training and education are essential for both practicing physicians and well as being a regular part of medical school curriculum. Training and education in pharmacogenomics must reach all of those who have a role in the delivery of the ways and means for personalized medicine.” Training is also required for industry scientists and regulatory reviewers.

Designing Preclinical Safety Evaluation Programs of Biopharmaceuticals

The goal of a preclinical development plan is to engineer a study design that will enable a seamless transitioning from discovery to the clinic and ultimately to a product approval as quickly as possible while limiting costs. However, by going too fast we risk missing critical steps, resulting in repeated processes and studies. It must be determined if there is a cost savings to taking the time and setting more realistic milestones in order to get it right the first time. Amgen’s Jeanine Bussiere stated, “Industry has learned some valuable lessons, such as, ‘A two-week toxicity study does not mean a project two weeks in duration,’ and ‘The FDA’s fast track does not mean that the FDA reads faster.’” Accelerating the clinical development program requires project teams that include representatives from upper management, and establishing processes early that will alleviate future bottlenecks.

There will, of course, be inevitable delays based on a variety of factors, including the availability of the test article, appropriate analytical methods, and relevant animal models (Table 1). For biotech-derived pharmaceuticals, additional factors are involved, including limited species cross-reactivity which necessitates alternative or novel preclinical programs (Table 2). In addition, changes to manufacturing process or route of administration may necessitate additional studies resulting in more time, effort and money, as well as higher risks of failing for technical, scientific or regulatory reasons. Different timing of key development decisions may be needed to facilitate clinical development and manage risk.

Table 1: Factors Impacting Study Delays

•    Test Article
    o    Availability
    o    Contract Manufacturing Organization
    o    Quantity, product stability, formulation

•    Analytical methods
    o    Sensitivity, specificity
    o    Ability to quantify test article
    o    Reagents

•    Relevant animal model(s)

•    Biomarkers of activity/safety

•    Experienced staff

•    Adequate funding

•    Quality of the data

•    Clinical plan

•    Regulatory path/expectations

•    Management

Table 2: Generalized Differences in Study Design For Conventional Drugs vs. Biopharmaceuticals

Conventional Pharmaceuticals

•    Previous examples

•    Historical database

•    Toxicity

•    Maximum tolerated dose

•    Species independent

•    Metabolized

•    Isomers

•    Specific mechanism

•    Short acting

•    Chronic daily dosing

•    Direct effects

•    Complex formulations

•    Oral route

•    Bioequivalence

Novel Biopharmaceuticals

•    Unique

•    Concurrent controls

•    Exaggerated activity

•    OBD

•    Species specific

•    Degraded

•    Secondary, tertiary or quaternary structures/labile bonds

•    Pleiotrophic mechanisms

•    Long acting

•    Intermittent dosing

•    Complex temporal relationships

•    Simple formulations

•    Injections

•    Comparability

The industry is experiencing an evolution with a changing regulatory climate and increased emphasis on the development of biological drugs. According to Dr. Bussiere, accelerated approval may result in TOO fast a track. “We all understand that drug development is money; however, we cannot risk an erosion of trust and must understand that in the consumer’s mind ‘risk sharing’ is equivalent to ‘risk aversion.’ The emphasis must continue to be safety!” she declared.

Predicting risk means determining if a product is safe and entails better ways to maximize benefit and improve the science of predicting toxicity. It involves the development of more effective risk communication and collaboration on improving the quality of pharmaceutical care.

The preclinical dilemma results from a sometimes schizophrenic use of data based on a belief in the efficacy but skepticism about toxicity due to the inefficient use of “proof of concept” models with the active dose and lack of safety endpoints. This approach is designed to satisfy a discipline rather than providing answers to questions for clinical decision-making. The plan for accelerating development involves moving away from the empirical studies ritual and toward informed, rational questioning and determining what issues should be addressed to evaluate safety concerns for the clinic.

Timelines for comparative safety studies between conventional and biological drugs involve significant differences. It is estimated that for a conventional drug the time to file an IND is approximately 10 months, while the timeline for a biologic is as long as 17 months. Much of the difference involves the lead time and cost for manufacturing, since the cost of the biologic being much higher ($1.6 – $2.6 million for 35 g versus $40K – $1.2 million for 1.3 kg). Similar differences can be seen in the safety testing support for Phase II, where the availability of biomarkers of safety/efficacy are key. In addition, reproductive studies are generally done in nonhuman primates which takes longer and is significantly more expensive. In the comparative preclinical timeline to support Phase III and approval the roles are switched with the biologic having some advantages due to the lack of need for carcinogenicity studies as well as no additional support needed for registration versus a 36-month support time for conventional drugs.

Rational science-based preclinical development is defined by data-driven, practical studies designed to obtain maximum information. By applying a modified approach based on an existing knowledge base, limitations and knowledge gaps are identified and new models can be used to answer questions about ongoing activities. Rational science-based preclinical development does pose practical challenges, such as timing of product availability, assay development and validation. For biotech-derived pharmaceuticals in particular, ultimate success is achieved by “de-risking” programs early through detailed, long-range planning, frequent review of progress, and early and frequent regulatory interactions.

The Exploratory IND

The Exploratory IND offers a unique opportunity to gain important information about a biologic in a clinical setting prior to traditional IND studies. According to FDA supervisory pharmacologist David Green this is an important time of change for the agency as it evolves to be more responsive and transparent. He commented, “The exploratory IND process is designed to encourage the identification of candidate drugs and biologics as well as improve the understanding of the basic science that underlies new therapeutic and diagnostic approaches.” The development of biologics relies on characterizing disease-related targets and selecting suitable molecular qualities to yield a potential candidate for clinical study. Developers may select and modify both pharmacodynamic and pharmacokinetic characteristics in their product with a reasonable expectation of its performance in vivo. Nevertheless, it is critically important to validate these working assumptions prior to initiating a traditional IND.

Although existing regulations allow for a great deal of flexibility for INDs, this flexibility was not often utilized, and the exploratory IND covers preclinical standards and clinical study parameters, as well as chemistry, manufacturing and controls standards. This is not drug development in the typical or traditional sense, but rather addresses the need to characterize new therapeutic targets, identify lead candidates to interact with a specific therapeutic target, perform ‘scouting’ studies of pharmacokinetics and/or pharmacodynamics in humans, and examine biodistribution using various imaging technologies. It is not intended to provide means of selecting a therapeutic dose nor to supplant a traditional IND process.

Upon completion of an exploratory IND, the application is withdrawn and if appropriate, new clinical studies may be initiated under a traditional IND that is supported by preclinical studies. Results of the Exploratory IND should be discussed with the agency after closing and prior to submission of a traditional IND. The process should emphasize and increase the contribution of science to the selection of drug candidates and promote efficient and effective use of resources. This could potentially increase the number and rate of therapeutics developed for patient use and remain consistent with the protection of human subjects. Implementation of this guidance is a vital part of the FDA’s commitment to improving the “Critical Path” for new medical products. The preclinical safety data required for Exploratory INDs will be designed to match the proposed clinical study and in general be less than conventional INDs. The reduction in safety data requirements will match the limited goals, duration and scope of the proposed clinical trials.

Table 3: Exploratory IND Clinical Studies of Pharmacokinetics or Imaging

•    Micro-dose concept for safety

•    1/100th of pharmacological dose and < 100 micrograms

•    PK information in human subjects

•    Study of imaging agents of specific targets

•    Not designed to induce pharmacological effects

•    Select compounds for further development

•    Given only once in clinical trial to healthy subjects

•    Supported by nonclinical, extended single-dose toxicity study

•    Single mammalian species if justified by metabolism data or in vitro pharmacodynamic effects

•    Routine genetic toxicity testing not needed

Studies of pharmacokinetics or imaging include a single dose study in relevant species with 14-day observation and interim sacrifice, full clinical and histopathology. Clinical trials are done to study pharmacological effects especially after repeated administration. The upper limit on clinical dose avoids increased risk inherent in traditional designs. For example, to support a repeat dose clinical study of as long as seven days, a two-week repeat dose nonclinical study and a toxicokinetic evaluation are needed.

Several issues remain to be clarified including a greater distinction between drugs and biological products as well as the continuing debate around relevant species and the relationship to ICH S6. The exploratory IND is another in a series of improvements with the objective for greater flexibility and scope in drug development. However, more definition of the relationship between exploratory INDs and conventional INDs must be examined as well as greater specification of the studies needed for clinical studies.

Considerations in Immunotoxicology

Although immunotoxicology (i.e., suppression or enhancement of immune response) has been an important topic for regulators, biologics have generally been exempt because many of these drugs are intended to affect the immune system. This is primarily an historical artifact, but does have foundation in scientific differences between protein drugs and small molecular weight compounds. Many biologics were originally developed to have immunomodulatory activity. Hence, what would be considered “immunotoxic” in the small molecule drug world would in fact be considered exaggerated pharmacodynamics (PD) for biologics. According to Ken Hastings, the associate director of pharmacology and toxicology at CDER, “The situation has become more complex as the line between small molecule drugs and biologics has become less defined, and adverse effects have been observed which are clearly not PD-related.”

Although ICH S8 contains an exemption for biologics, the important principles in this guidance can be useful in evaluating the safety of protein drugs. A distinction must also be drawn between unintended and intended effects that impact on the immune system as an important part of any toxicology study. The FDA guidance document addresses several topics including immunosuppression, immunogenicity, hypersensitivity, autoimmunity and adverse immunostimulation.

Table 4: Considerations in Immunotoxicity Evaluation of Biotherapeutics

•    Topical drug

•    Inhalation drug

•    Evidence of immunosuppression

•    Drug for HIV infection

•    Use in pregnancy/evidence of immunosuppression

•    Immune system accumulation

•    None of the above?

Table 5: Evidence of Immunosuppression in Nonclinical Toxicity Studies

•    Evidence of myelosuppression

•    Alterations in immune system organ weights and/or histology

•    Increased incidence of infections

•    Increased incidence of tumors

•    Decreased serum immunoglobulin levels

The goals of the studies are to determine dermal and respiratory sensitizing potential, immune system effects in F1 pups, and possible immune effects. A distinction must be drawn between unintended (adverse) and intended (pharmacodynamic) immunosuppressive effects. Important points to consider are the reliance on results of standard toxicity studies, stand-alone functional assays, and need for enhanced histopathology (e.g., draining lymphoid tissue). Where depletion or hyperplasia of lymph nodes and spleen are observed, a better description of cell types affected and hematologic effects needs to be evaluated. Claims that immunosuppression was due to stress must be supported with comprehensive data and functional assays are recommended if effects are seen in standard tests.

Several EMEA/CPMP guidelines include incorporation of immunotoxicity testing in 28-day rodent studies including hypersensitivity and photoallergy testing. One of two sets of data can be used. The first includes hematology, lymph-oid weights and histology, bone marrow cellularity, immunophenotyping, and NK cell activity. The second is an evaluation of the primary antibody response to a T-dependent antigen. These studies can be conducted prior to or concurrent with Phase II studies

Dr. Hastings noted it is a goal to harmonize these different issues by inclusion in ICH S8. “The efforts serve several important purposes. While the CPMP Note for Guidance essentially calls for mandatory testing for unintended immunosuppression, the FDA has essentially taken a cause-for-concern approach,” he said, after much debate, the issue was accepted as an ICH topic in 2003 and a Step 4 document was finalized in 2005. This document includes recommendations for nonclinical testing approaches to identify compounds that have the potential to be immunotoxic and recommends a weight-of-evidence decision making approach. The guidelines make no recommendations about testing for drug-specific immune-mediated hypersensitivity and other tests could be recommended. However, signs of enhancement as well as lymphocytosis or inflammation should be evaluated. There are no specific recommendations for follow-up testing.

The ICH guidance on immunotoxicity is an important step. Although specific immunotoxicity testing is not mandated, if weight-of-evidence indicates need, regulatory agencies have the authority to request studies. Claims that signs of immunotoxicity are due to stress effects must be compelling. Drug allergy and autoimmunity issues cannot be addressed without validated methods.

Preparing for the Dynamics of the Future

Drug delivery is becoming more and more critical for biologics due to the need to deliver small amounts to target in more potent forms. A significant amount of training and expertise is needed to address biologics and significant advances in the expertise and methodologies needed for comprehensive safety evaluation occur at a dizzying pace. Regulators are dependent on industry for taking a leading role in the development of tools and data needed to accelerate the testing and approval of these important therapies.

Acknowledgement

This article is a summarized report of a series of presentations at a symposium held in November 2005. The authors gratefully acknowledge the participation and expertise of the speakers quoted in the article for their insight on the topics covered in this manuscript.

Steve Barkyoumb is vice president, Toxicology North America, at Covance. He can be reached at steve.barkyoumb@covance.com.

Chris Springall is vice president, Toxicology Europe, at Covance. He can be reached at christopher.springall@covance.com.

Friedhelm Vogel is managing director, Covance Munster Germany. He can be reached at friedhelm.vogel@covance.com.

Richard Crowley is senior science writer at Covance. He can be reached at rick.crowley@covance.com.