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MOU 225-94-3001

Memorandum of Understanding

Between
The United States Department of Health and Human Services
National Institutes of Health
National Institute on Aging
Laboratory of Neurosciences 

and

The United States Department of Health and Human Services
Food and Drug Administration

 

I. PURPOSE

The Food and Drug Administration (FDA) and Laboratory of Neurosciences (LNS), National Institute on Aging, National Institutes of Health participated in an Interagency Agreement (FDA 224-91-1301) for FY 91 and FY 92 entitled, "The Development of a Practical Method for Detecting Neurotoxicity Based on the Autoradiographic Measurement of Cellular Metabolic Markers." The purpose of the proposed Memorandum of Understanding (MOU) is to maintain a relationship between the FDA and LNS so that 1) experiments in progress can continue to completion 2) accumulated data can be collated, examined with statistics and submitted in manuscript form for publication in peer-reviewed journals and 3) follow up experiments can be planned and carried out. The outcome of these studies are of mutual interest to the FDA and LNS. Continuing the relationship between the participants provides a cost effective and time efficient way to bring this to fruition.

 

II. BACKGROUND

THE NEED TO DEVELOP A PRACTICAL SCREEN FOR NEUROTOXICITY WITH WIDE REGULATORY APPLICABILITY

 

The neurosciences represent one of the most rapidly developing areas of the biological sciences. Drug development in this area is characterized by rapid commercial application of discoveries, but the application of new methods and discoveries to the development and validation of new approaches for detecting and assessing neurotoxicity has lagged. The application of new developments, methods and technology in the medical sciences to answer toxicological questions of regulatory significance is within major F.D.A's mission.

 

Increasing public concern about the neurotoxicity of drugs and environmental agents and the adequacy of currently applied toxicological testing requirements has resulted in the initiation of a Congressional review of Federal research and regulatory programs by the Office of Technology Assessment (OTA). A workshop was sponsored by the OTA in April of 1989 and was attended by representatives of all agencies that deal with neurotoxicologic issues. The proceedings of this workshop will be incorporated into a report to congress that is expected to be released in mid-April 1990. This report is expected to include proposals and options for coordinating and expanding Federal neurotoxicity testing.

 

LIMITATIONS OF CURRENT NEUROTOXICITY TESTING

 

Neurotoxicity has been defined as an adverse effect on the structure or function of the nervous system. There are several approaches to the assessment of neurotoxicity. These can be generally categorized into three groups; behavioral assessments, morphological assessments (neurohistopathology), and biochemical assessments (measurements of altered cellular metabolism and function). Current preclinical neurotoxicity testing generally relies on the detection of behavioral abnormalities and/or the appearance of overt histopathological lesions in nerve tissue.

Animal behavioral studies are most effective in detecting neurotoxicity which adversely affect well defined and easily detectable parameters such as survival, motor function, aggression, feeding, grooming, reproductive and maternal behaviors. It is likely however, that a majority of the effects on the central nervous system are silent and produce changes in functions that result in subtle alterations in parameters such as emotion, cognition, temperament, or mood that cannot be adequately evaluated in animals and may not be easily identified in human studies. There are examples of lesions of the brain that produce little or no overt signs in animals or man.

 

Histological methods are effective in detecting neurotoxicity only when lesions of the nervous system are extensive enough and of a nature that can be detected by the staining methods employed. The number of sections analyzed and the availability of skilled neuropathologists are limiting factors in the effectiveness of this method. Behavioral testing and neurohistopathology are approaches that are labor intensive, require specialized skills of observation and training and are more influenced by subjective variability than are biochemical methods.

 

The application of biochemical markers for neurotoxicity is an ongoing area of research both within federal agencies and in the academic community. Several specific biochemical markers (eg. changes in enzyme activity, protein phosphorylation, or cell markers such as GFAP for reactive astrocytes) have been examined but have proven useful for detecting only selected types of neurotoxicity. Other biochemical assessments that measure brain function as correlated with metabolism (as described below), have not been rigorously tested.

 

THE OPPORTUNITY FOR FDA LEADERSHIP IN DEVELOPING AND VALIDATING NEW METHODS FOR ASSESSING THE NEUROTOXICITY OF DRUGS, FOOD ADDITIVES OR ENVIRONMENTAL AGENTS.

 

There is general agreement among all agencies dealing with the regulation of toxic substances and the academic community that current methods and criteria for detecting and assessing neurotoxicity are inadequate. What is needed is a biochemical method that can detect a functional alteration in neurons that is associated with a pathological change in neuronal function. Alterations in cellular processes such as energy metabolism, membrane integrity or protein metabolism are associated with neuronal damage and death and may be appropriate markers of neurotoxicity. A practical method should be relatively simple to carry out at reasonable cost with good predictive ability. An ideal method should also have the potential to be adapted to human.

 

There is also a need for better criteria for assessing potentially adverse CNS effects of drugs and other substances, especially those that are not intended or expected to act on the CNS. This is especially urgent for AIDS drugs and cancer chemotherapeutic agents.

 

BRAIN 2-DEOXYGLUCOSE, FATTY ACID AND AMINO ACID METABOLISM AS FUNCTIONAL ASSESSMENTS OF NEUROTOXICITY

 

Degenerative changes in the neuron appear to be associated with alterations in energy metabolism, protein metabolism and fatty acid metabolism. Alterations in the status of these parameters can now be assessed in vivo by measuring the uptake of 2-deoxglucose, isoleucine and fatty acids such as arachidonic and palmitic acid. These methods are well established in the literature, and are known to be capable of detecting neurotoxicity.

The best characterized of these biochemical-functional assessments is the 2-deoxyglucose method originally described by Sokoloff in 1977 and used to examine regional-cerebral energy consumption. In combination with quantitative autoradiography, this approach has been successfully applied to examine regionally selective changes in brain function in vivo in response to pharmacologically active agents and neurotoxic substances. For example, selective alterations in glucose metabolism in brain excitotoxin kainic acid as well with PCP, MK801, and carboxypiperazine propylphosphonic acid (CPP) have been reported. In combination with positron emission tomography (PET), the 2-deoxyglucose method has seen wide application in humans.

 

More recently, two methods related in principle to the 2-deoxyglucose method have been described which focus on the rates of synthesis of specific functional cellular elements. The radiolabelled amino acid (leucine) technique has been used to monitor regional changes in brain protein synthesis occurring during development and or as a result of pathologic insult. The radiolabelled fatty acid (palmitic and arachidonic acid) method, has been used to detect regionally specific changes in phospholipids synthesis associated with development, a variety of reversible and non-reversible pathologic insults and neuropharmacological stimulation. These more recent methods, while not measuring the global parameter of energy metabolism, measure metabolic rates for proteins and phospholipids. Their ability to report on synthetic rates of structural elements of cells, which would be expected to be altered by reversible or irreversible morphologic changes caused by a neurotoxicant, may prove more informative than global measures. Like the 2-deoxyglucose method, both procedures are amenable to use in humans.

 

III. SUBSTANCE OF THE AGREEMENT

Nathan M. Appel, Ph.D. (Biologist, FDA/CDER/ORR/DRT) and Robert L. Scott B.A. (Biologist, FDA/CDER/ORR/DRT) will collaborate on experiments with LNS as detailed below. Technical assistance, experimental animals, instrumentation and supplies required to perform the experiments will be provided by LNS. Samples resulting from experiments will be analyzed either at LNS or the FDA's Module 1 Laboratory Facility (Mod 1), Laurel, MD as circumstances dictate. Similarly, data analysis will be performed at both sites.

 

DESCRIPTION OF EXPERIMENTS

 

Animals will be treated systemically with neurotoxicants or vehicle and prepared for radiotracer incorporation. Studies use awake or anesthetized rats which are partially restrained on wooden blocks after the surgical implantation of femoral arterial and venous catheters. A radiolabeled fatty acid, 2-deoxyglucose, or leucine is injected intravenously, and timed arterial samples are withdrawn until the animal is killed. Brain radioactivity is determined by quantitative autoradiography on frozen 20 micron sections. Histological analysis of lesion development will be performed on companion brain sections, with radioactivity in remaining tissue sections determined by liquid scintillation. This later determination of radioactive probe incorporation can be used to calculate total uptake of probe into stable brain compartments. Brain radioactivity in specific stable brain compartments is determined from extraction fractionation and quantitation of radioactivity using a Berthold TLC radioactivity scanner or liquid scintillation procedures. Mathematical modeling and curve fitting are employed to calculate rates of tracer incorporation in specific brain regions based on autoradiographic data using data from systemic exposure to probe. Attempts will be made to simplify this regional quantitative analysis by determining regional relative rates of incorporation of probe by normalizing to total brain incorporation calculated by autoradiographic or biochemical analysis. These relative rates of incorporation will be compared with the absolute rates of incorporation in an assessment of their efficiency for detecting changes in metabolism associated with neuropathology.

 

If the normalization procedures prove successful in allowing detection of neurotoxic lesions, attempts will be made to develop a simplified assay system using the mouse. Use of the mouse will allow tail vein administration of the probes. This will eliminate the necessity of surgery for venous and arterial cannulation and make unnecessary the use of restraint currently required when using the rat as an experimental animal. The use of a smaller experimental animal also reduces cost significantly by reducing the quantity of isotope required, in addition to reducing animal cost and space necessary to house treated animals. If feasible, this approach may result in a greatly simplified method for use in drug and chemical toxicity screening. Development of the mouse model will necessitate extensive documentation of the neurotoxic effects of standard neurotoxins, since many reference neurotoxins have not been tested in the mouse.

 

THE FDA SHALL allow Nathan M. Appel, Ph.D. (Biologist, FDA/CDER/ORR/DRT) and Robert L. Scott, B.A. (Biologist, FDA/CDER/ORR/DRT) to perform experiments detailed above. Experiments will be performed at LNS or MOD 1. Samples resulting from experiments will be processed either at LNS or MOD 1 as circumstances dictate. Similarly, data analysis will be performed at either site.

 

IV. Participating Parties

Laboratory of Neurosciences (LNS)
National Institute on Aging, National Institutes of Health
Building 10, Room 8C-10S
Bethesda, MD 20892-0010

Division of Research and Testing
Office of Research Resources
Center for Drug Evaluation and Research
Food and Drug Administration
Module 1 Laboratory Facility
8301 Muirkirk Road
Laurel, MD 20708

 

V. Liaison Officers

For the Laboratory of Neurosciences:

Stanley I. Rappoport, M.D.
National Institute on Aging
National Institutes of Health
Building 10, Room 6C-10S
Bethesda, MD 20892

 

For the Food and Drug Administration:

Nathan M. Appel, Ph.D.
Division of Research and Testing, HFD-420
Food and Drug Administration
Module 1 Laboratory Facility
8301 Muirkirk Road
Laurel, MD 20708
Telephone (301) 594-5027

 

Joseph F. Contrera, Ph.D.
Office of Research Resources, HFD-400
Food and Drug Administration
5600 Fishers Lane
Rockville, MD 20857
Telephone (301) 443-4750

 

Joseph J. DeGeorge, Ph.D.
Division of Oncology & Pulmonary Drug Products
Office of Drug Evaluation I, HFD-150
Food and Drug Administration
5600 Fishers Lane
Rockville, MD 20857
Telephone (301) 295-9135

 

VI. Period of Agreement

This agreement becomes effective upon acceptance by both parties, and will continue in effect indefinitely. It may be modified by mutual written consent or terminated by either party upon a 60 day advance written notice to the other party.

Approved and Accepted
for the Laboratory of Neurosciences

Signed by: Scientific Director
National Institute on Aging

Date: January 4, 1994

Approved and Accepted
for the Food and Drug Administration

Signed by: Assistant Director, ORR

Date: January 4, 1994