The Walter Reed Army Institute of Research
WRAIR
Colonel Daniel L. Jarboe, VC, Commander and Institute Director

           Bearing the name of one of the US Army's greatest medical investigators and practicing physicians, Major, Walter Reed, MC, MD, the Walter Reed Army Institute of Research (WRAIR) continues in his tradition of providing the very best medical research available. As Walter Reed conquered typhoid fever for the US Army and then "Yellow Fever" for the Army and the world, WRAIR today is providing that same dedicated, exceptionally professional service to an even wider variety of medical problems that confront the armed forces and the civilian community. The WRAIR's mission encompasses many facets and this article will give the highlights of the Division of Biochemistry. In future issues, ASA hopes to provide detailed information on other very important Divisions of the WRAIR.
           The Division of Biochemistry: Division Director
Bhupendra P. Doctor, Ph.D.
           Chief, Department of Membrane Biochemistry:
Carl R. Alving M.D.
           Chief, Department of Molecular Pharmacology:
Ashima Saxena Ph.D.
           Chief, Department of Biochemical Pharmacology:
Richard K. Gordon, Ph.D.

           Heading up one of WRAIRs most active divisions is an original and very valued member of the CBMTS community, Dr. Bhupendra Doctor, the Director of the Division of Biochemistry. Continuing in the tradition of Walter Reed, this Division has been responsible for many exceptionally important advances in medical research for the soldier. Additionally the Division of Biochemistry is continually involved in on-going medical research projects, which will not only enhance the well being of the soldier, who must often survive in hostile, disease-ridden and health threatening environments of chemical and biological warfare agents, but will enhance the medical well being of the civilian community as well. Many of these projects are developed both within the Departments and in collaboration with scientific institutes worldwide.

           Dr. Ashima Saxena, also a CBMTS member, heads up the Department of Molecular Pharmacology. This department is responsible for developing and pursuing basic and applied scientific research that is vital to the successful completion of the vast array of exceptionally important projects related to chemical warfare agent toxicity.
            The Department is conducting the following research projects related to providing protection to soldiers from organophosphate chemical nerve agent (OP) poisoning. The main objective is to develop a pretreatment regimen that does not cause any performance decrement and at the same time totally protects the life of the soldier:
            (1) Adaptation of the bioscavenger approach to this problem has been demonstrated by us to be highly effective. Human serum butyrylcholinesterase (Hu BChE) is judged and proven to be the best overall, natural, human compatible bioscavenger. The group is actively involved in developing a commercial scale production procedure for the purification of Hu BChE from Cohn fraction IV, a discarded fraction of pooled human plasma.
            (2) Determine the safety and efficacy of purified Hu BChE in mice, guinea pigs and non-human primates: Toxicokinetics and pharmacokinetics studies are being performed at the TNO Laboratories in the Netherlands
            (3) Perform neutron scattering studies of Hu BChE in collaboration with scientists in Grenoble (Cressa & IBS laboratories) France and Rehovot (Weizmann Institute) Israel, to learn more about the active site (the site that binds OPs) with the goal of improving it.
            (4) Oximes (nucleophiles) reactivate OP-inactivated cholinesterases by binding to them and releasing functional cholinesterase (thereby serving as a post-exposure therapy); a phosphorylated oxime intermediate is also formed. Since this complex is still toxic, it must be broken down by hydrolases. The Department is investigating the following: The role of amino acid residues in the active site of various cholinesterases in the reactivation of OP-inhibited mouse AChE and Hu BChE by oximes. The use of OP hydrolases in eliminating phosphoryl oximes formed during reactivation.
            (5) Develop a gene delivery system (by inhalation) using liposomes or adenoviral vectors to deliver the BChE or AChE gene into the lungs, as a long-term prophylactic strategy.
            (6) Evaluate various FDA-approved drugs such as Aricept (E2020) and galanthamine as replacements for pyridostigmine. Pyridostigmine binds reversibly to a portion of Hu AChE and thus protects it from binding to OPs, thus reserving this protected Hu AChE as a reservoir of functional enzyme. Other reversible ChE inhibitors such as Huperzine A and its analogs are also investigated for this purpose.

           The Department of Biochemical Pharmacology, headed by Dr. Richard Gordon, a CBMTS member, is involved in many exciting and productive projects that are considered directed research for the soldier - to provide the soldier with protection against chemical and biological warfare agents. These projects include:
           (1) Developing enzyme immobilized organophosphate decontamination and detoxifying sponges for personal use by the soldier in the field without an additional weight burden. This project involves purifying and immobilizing multiple OP hydrolyzing enzymes into sponges, packaging the sponges in a form to wipe skin and other surfaces, and evaluating additives to remove/decontaminate/detoxify OPs from biological surfaces. Now underway are studies to evaluate additives to ensure detoxification of other chemical warfare agents such as vesicants (mustard, HD).
           (2) Develop biosensors for detecting organophosphates and HD in most environments, including air, dirt, or water, which the current kits cannot, and development of biosensors for OPs that can discriminate in the field which OP is present. The project involves the development of multiple immobilized enzyme biosensors for differential detection and discrimination of OPs. A patent covering this technology was issued this year.
           (3) Develop high-throughput robotic cholinesterase assay techniques to measure both AChE and BChE simultaneously from as little as 10 uL of human blood. This technology is being modified to be suitable for hand-held devices for field use.An important aspect of the method is that it could provide a standardized laboratory protocol to determine the level of cholinesterases in human and animal blood, and indicate diseases associated with cholinergic enzymes.
           (4) Evaluate pharmaceutical agents to be administered pre- and post-nerve agent exposureto rescue potentially salvageable neurons and/or mitigate the resulting neurobehavioral functional impairment seen after recovery from acute nerve agent exposure. Some examples of compounds being evaluated are Huperzine A and its analogs. The pharmacodynamics and pharmacokinetics of Huperzine A in vivo are being determined. Huperzine is obtained from a Chinese herb, and selectively and reversibly binds AChE over BChE. The WRAIR biochemists have shown that Huperzine has dual protective roles, first sparing AChE from OPs and second, it protects against neurotoxicity. In the latter case, Huperzine blocks (occludes) the ion channel of overstimulated glutamate receptors, which otherwise would result in cell energy depletion and neuronal toxicity.
           (5) Develop novel analytical techniques using sophisticated equipment to quantify low levels of pyridostigmine bromide, atropine, scopolamine, and Huperzine A and its analogs in various biological samples including human blood, urine, and tissues. As such, a GLP lab meeting FDA guidelines has been established so that the obtained clinical data can support FDA INDs.
           (6) The lab group is also developing methods to protect against biological warfare agents, specifically botulinum toxin. The goal is to develop peptide therapeutics for botulism that is effective against its multiple serotypes and tetanus toxin. Peptide inhibitors (patents pending) have been designed. Now, the researchers are exploring peptide delivery systems for use in field operations to ameliorate the effects of these toxins.

           The Department of Membrane Biochemistry is headed by Carl R. Alving M.D. (COL, U.S. Army Medical Corps, Ret.). The development of the technique of needle-free transcutaneous immunization (TCI) by skin patch is counted among many dramatic medical advances that have been initiated and developed over the years by the Department of Membrane Biochemistry. TCI is a dual-use technology that has been transitioned to commercial development under the Technology Transfer Act of 1986 and through licensing of the technology to Iomai Corporation of Gaithersburg, Maryland. TCI was co-invented by Dr. Alving while he was on active duty, and Dr. (formerly MAJ) Gregory Glenn, a pediatrician who was also on active duty in Membrane Biochemistry and who is now Chief Scientific Officer for Iomai. In collaborations between WRAIR and Iomai, a skin patch vaccine utilizing TCI technology has been tested at WRAIR in a phase I clinical trial in volunteers for application to a traveler's diarrhea vaccine against heat-labile E. coli enterotoxin. Iomai Corporation has conducted civilian phase I/IIa trials with a tetanus toxoid vaccine. The Department is also developing the TCI technology for several applications, including skin patch vaccines for anthrax, Ebola virus, HIV, malaria and others. It is anticipated that the TCI strategy, which promotes both potent parenteral and mucosal immunity, will have numerous vaccine applications.
           Because of its expertise in adjuvants, the Department of Membrane Biochemistry was requested to address the role, if any, of antibodies to a commonly used oil adjuvant, squalene, in the development of pathological symptoms in Gulf War veterans. This issue has been widely raised in the civilian community and in Congress as a potential detrimental effect of the anthrax vaccine. This has generated controversy, because many scientists consider the scientific basis of whether anti-squalene antibodies are induced by the anthrax vaccine, and of whether such antibodies would have pathological effects, questionable. However, because of wide public interest, the Membrane Biochemistry, at the request of DOD, is conducting research to apply rational scientific principles to this question. As part of the process of developing high throughput assays for testing of antibodies to squalene in humans, monoclonal antibodies to squalene have been created as positive controls. With the new assay developed by the department, natural antibodies to squalene unexpectedly have been discovered in healthy normal humans, and the testing of cohorts of Gulf War veterans who have complained of Gulf War illnesses is about to start. ASA will publish updated information in this area as the information is released.
           Membrane Biochemistry is a leader in the field of innate immunity, the immunological mechanisms that form the first line of defense against foreign invaders. Their research has demonstrated the strong involvement of the complement system as a major complicating element in fluid resuscitation under conditions of acute trauma. Measures to counteract the adverse effects of complement, particularly through the development of inhibitors, have been evolved and tested in animal models by the department. The department, via the Vaccine and Immunology Research Institute (VIRI), also organizes and manages the WRAIR-sponsored National Symposium on Basic Aspects of Vaccines. The eighth meeting in this symposia series was completed in May, 2002.

           There are many other projects being conducted by members of the Biochemistry Division in coordination with other Divisions at WRAIR and with other primary organizations and institutes within the US Army Medical Research and Materiel Command (MRMC).

           Dr. Matthew Clark, who gave an excellent presentation at this last CBMTS, is conducting research in the Mouse Neurobehavioral Assessment Laboratory, Division of Neurosciences, at WRAIR for the purpose of developing an animal model capable of assessing the neurobehavioral effects of chemical warfare agent exposure and treatment. Using a portion of this behavioral test battery, Dr. Clark and his team have been able to assess the effect of repeated exposure to low levels of the CW agent VX. Additionally, Dr. Clark is heading up two other research teams investigating the effects of nerve agents and the safety of nerve agent prophylactics in a non-human primate model.

           In one of the many joint research projects between WRAIR's Biochemistry Division, Neuroscience Division and the Institute of Chemical Defense (ICD), Dr. Sally Anderson and the WRAIR team, Maj. Maurice Sipos and the ICD team are working on the project "Effects of subacute exposure to low doses of chemical warfare nerve agents on esterases and cellular energy systems in the brain". This project's preliminary goals are to demonstrate the relationship between cholinesterase inhibition in different brain regions and to evaluate the use of blood cholinesterase inhibition as a predictor of brain inhibition. The long-term focus is on compromise of brain energy systems by chronic low dose exposure.

           ASA wishes to thank Dr. Bhupendra Doctor, Director, Biochemistry Division and Colonel Daniel Jarboe, VC, Commander and Director, WRAIR for their hospitality and for permitting ASA to visit and to further understand the importance and vitality of their dynamic mission of medical research for the soldier. Thanks are also due to Dr. Ronald Goor, WRAIR's Research Marketing and Policy Director, for his help and assistance in putting this article together.

           Dr. Doctor has been mentioned in this Newsletter many times including the announcement and picture of him receiving the "Distinguished Executive, Presidential Rank Award in 1997". Dr. Doctor had previously received the Meritorious Executive, Presidential Rank Award in 1986.

Picture Credits: Mr Phil Collins.

ASA Note: 'Doc' - you and your Biochem Division are doing great work. We wish you continued success.