PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`(51) International Patent Classification 7 :
`COSG 18/00
`
`
`
`(21) International Application Number:
`PCT/USOO/11072
`
`(22) International Filing Date:
`26 April 2000 (26.04.00)
`
`
`
`
`A2
`
`(11) International Publication Number:
`
`WO 00/64957
`
`(43) International Publication Date:
`
`2 November 2000 (02.l LOO)
`
`(81) Designated States: AE, AG, AL, AM, AT, AU, AZ, BA, BB,
`BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM,
`DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL,
`IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU,
`LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT,
`RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ,
`UA, UG, US, UZ, VN, YU, ZA, ZW, ARIPO patent (GH,
`GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR,
`IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`(30) Priority Data:
`
`
`60/ 1 30,988
`26 April 1999 (26.04.99)
`US
`
`
`
`
`(71) Applicant (for all designated States except US): US. ARMY
`
`
`MEDICAL RESEARCH AND MATERIAL COMMAND
`[US/US]; Department of Army, Command Judge Advocate
`
`
`Office, 504 Scott Street, Fort Detrick, MD 21702—5012
`(US).
`
`
`(71)(72) Applicants and Inventors:
`GORDON, Richard, K.
`[—/US]; 9024 Willow Valley Drive, Potomac, MD 20854
`(US). DOCTOR, Bhupendra, P. [—/US]; 10613 Great Arbor
`Drive, Potomac, MD 20854 (US).
`
`(74) Agents: WISEMAN, Thomas, G. et al.; Morrison & Foerster
`LLP, 2000 Pennsylvania Avenue, N.W., Washington, DC
`20006—1888 (US).
`
`
`
`
`
`
`
`
`
`
`
`
`
`(54) Title: DIFFERENTIALLY ACTING OP DETOXIFYING SPONGES
`
`
`
`
`
`Final product: FBS-AChE sponge
`
`(57) Abstract
`
`A material comprising a porous support and a plurality of enzymes for the removal, decontamination or neutralization of hazardous
`
`
`chemicals such as OP compounds is disclosed. The material may be used on a variety of surfaces, including natural, synthetic and biological
`
`surfaces such as skin and other delicate membranes. Also disclosed is a process of making the material, kits and various methods and
`reactivation devices for reactivating the enzymatic activity of the material.
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Zimbabwe
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Buigaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`C6te d‘lvoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`[E
`[L
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`N0
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`
`

`

`WO 00/64957
`
`‘
`
`PCT/U800/11072
`
`DIFFERENTIALLY ACTING OP DETOXIFYING SPONGES
`
`Weld
`
`This invention relates to materials, compositions, kits and methods for neutralizing,
`
`detoxifying or decontaminating equipment and/or personnel exposed to organophosphorus and
`
`organosulfur compounds.
`
`Background of the Invention
`
`Methods for decontamination, neutralization and removal of chemicals, such as
`
`organophosphorus and organosulfiar (OP) compounds, herbicides and insecticides, are known
`
`in the art. However, the compositions and devices utilized in the prior art methods have
`
`undesirable properties, such as corrosiveness, flammability, toxicity, difficulty in making and
`
`storing, and limited shelf-life.
`
`For example, DSZ, a standard decontamination agent, comprises 70%
`
`diethylenetriamine, 28% ethylene glycol monomethyl ether, and 2% NaOH by weight.
`
`Although D82 is effective, it is corrosive upon exposure to air. D52 and any matter resulting
`
`from its use is classified and regulated as hazardous material. After an application, the D82
`
`must stand for 30 minutes before rinsing the treated area with water. Additionally, DSZ
`
`comprises a teratogen.
`
`Some decontamination methods employ hypochlorite formulations which are corrosive
`
`and toxic and injure humans and sensitive tissues such as eyes. Other methods comprise
`
`incinerating the contaminated material and utilizing carbon filters to absorb the residual
`
`chemicals. Yet other methods utilize polymer beads or microemulsions which absorb the
`
`chemical and must be rinsed away. These methods are inherently dangerous, expensive and
`
`generate hazardous waste. Furthermore, as many of these compositions and compounds
`
`utilized degrade upon exposure to water and carbon dioxide, these compositions and
`
`compounds must be used the same day they are made.
`
`Some in vivo methods employ cholinesterases in the presence of nucleophilic oximes to
`
`detoxify OP compounds. This enzyme bioscavenger approach is effective against a variety of
`
`OP compounds in rodents and nonhuman primates. For example, pretreatment of rhesus
`
`monkeys with fetal bovine serum acetylcholinesterase (FBS-AChE) or horse serum
`
`butyrylcholinesterase (Eq-BChE) confers protection against up to 5 LD50 of soman, a highly-
`
`toxic OP nerve agent. Although, the use of an enzyme as a single pretreatment drug for OP
`
`

`

`WO 00/64957
`
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`' 2 '
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`PCT/USOO/11072
`
`toxicity is sufficient to provide complete protection to an individual subject, a relatively large
`
`(stoichiometric) amount of the enzyme is required to neutralize the OP compound in viva.
`
`Therefore, OP/enzyme stoichiometry is increased by combining enzyme pretreatment with
`
`oxime reactivation so that the catalytic activity of OP inhibited FBS-AChE is rapidly and
`
`continuously restored, and the OP compound is detoxified.
`
`Clearly, a need for better methods and devices for neutralizing, detoxifying,
`
`decontaminating and cleaning materials, equipment and personnel exposed to OP compounds
`exists.
`
`Thus, OP detoxifying compounds, devices and methods thereof, which allow the safe,
`
`effective and convenient detoxification and quantitative and qualitative determination of highly
`
`toxic compounds not possible by the prior art, have been developed. These environmentally
`
`friendly compounds, devices and methods are disclosed hereinbelow.
`
`Summary of the Invention
`
`The present invention provides materials, compositions, kits and methods for
`
`neutralizing, detoxifying or decontaminating equipment and/or personnel exposed to OP
`
`compounds.
`
`In one embodiment, the invention relates to a material comprising a mixture of
`
`enzymes and substrates for the removal, decontamination and neutralization of OP compounds
`
`including those directed against humans. The mixture of enzymes utilized comprises
`
`cholinesterases (ChEs) and/or OP hydrolases and reactivators, such as OP reacting
`
`compounds such as certain oximes like PH-6 and mono—bisquartemary oximes such as 2—PAM.
`
`The material may comprise a flexible or rigid porous support. The porous support may
`
`be a polyurethane matrix or equivalent.
`
`For example, the porous support may be a flexible sponge-like substance or like
`
`material, wherein the enzymes are secured by immobilization. Depending on the polyurethane
`
`prepolymer or substrate utilized, porous supports of varying degrees of flexibility and porosity
`
`may be obtained. The porous support may be formed into various shapes, sizes and densities,
`
`depending on need and the shape of the mold. For example, the porous support may be
`
`formed into a typical household sponge or a towelette. The preferred dimensions of the
`
`sponge are l” x 2” x 8” to 2” x 4” x 8”. The preferred dimensions of the towelette are 4” x 4”
`
`x 0.25” to 4” x 4” x 0.03125” to 14” x 14” x 0.0625”. However, during large-scale synthesis,
`
`the dimensions of the initial immobilized enzyme product might be large. For example,
`
`

`

`wo 00/64957
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`PCT/US00/11072
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`approximately 4 feet by 8 feet rolls could be produced and sized as appropriate and described
`
`above.
`
`The sponge-like support would be preferable for use on surfaces, including natural,
`
`synthetic and biological surfaces such as equipment, laboratory hardware, devices, skin and
`
`other delicate membranes, where decontamination of a rough or irregular surface is desired or
`
`where the prior art decontamination materials are incompatible with human tissue. For
`
`example, the materials may be used to clean and decontaminate wounds as it is non-toxic and
`
`the immobilized enzymes will not leach into a wound. Therefore, the sponges could be used
`
`to decontaminate civilians contaminated by a terrorist attack at a public event.
`
`If an object and/or area to be neutralized or decontaminated comprises cracks,
`
`crevices, porous or uneven surfaces, a foam-like support is suitable. Application of small
`
`quantities may be done with a spray-bottle or spray can with an appropriate nozzle. Further,
`
`foam may be selected so that it can be dispensed into the opening of sensitive equipment or an
`
`orifice of a subject, such as the ear canal. If a large area is contaminated, an apparatus that
`
`dispenses a large quantity of foam may be utilized.
`
`The foam-like support may dissipate after a period of time like shaving cream or it may
`
`cure into a stable and flexible sponge-like support. The dissipating foam may be applied on
`
`living subjects. The foam, which cures, may be applied around an object and contain the
`
`contamination within the foam. Once the foam cures, the object may be handled and moved
`
`without fiirther exposure to the hazardous chemical.
`
`When necessary, the material may also comprise a rigid and porous support. The rigid
`
`material can be ground into a powder and added to lotions, soaps and other liquids for
`
`application. Likewise, the flexible material, supra, may be appropriately treated to render it
`
`suitable for use in lotions, soaps and other liquids.
`
`The material may also be in the form of a filter for neutralizing, detoxifying or
`
`decontaminating gases such as air. Additionally, the material may be in a form suitable for use
`
`as clothing or linings of clothing. Furthermore, the material may be used to decontaminate
`
`water by placing the material in water and then removing it from the water.
`
`In another embodiment, the material can be color-coded according to the specific
`
`substance it may neutralize, detoxify or decontaminate. The color or color scheme could be
`
`selected to indicate enzymatic concentration, activity and/or remaining shelf—life or range
`thereof.
`
`

`

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`
`The materials of the invention may be placed in containers to complete
`
`decontamination of the OP compounds on the materials.
`
`Other embodiments include the methods of using the instant materials for the
`
`quantitative or qualitative determination of hazardous compounds such as OP compounds.
`
`As disclosed herein, one of ordinary skill in the art will appreciate the various materials
`
`and their uses as contemplated by the inventors. All of these forms may be appropriately
`
`combined with carbon for further absorption of OP compounds. The carbon may be
`
`embedded or incorporated within the porous support of the material or the carbon may be a
`
`layer, filter or other to be used in conjunction with the material. Additionally, a slow release
`
`form, such as a dry capsule, pellet, liposome or other, of a reactivating compound such as EH—
`
`6 may be embedded or incorporated within the porous support of the material.
`
`A preferred embodiment of the invention comprises a material wherein AChE and/or
`
`BChE are simultaneously immobilized with OP hydrolases on or within the porous support
`
`during synthesis of the material. Preferably, the enzymes are immobilized through covalent
`
`linkages. The enzymes may be of prokaryotic or eukaryotic origin. These enzymes may be
`
`recombinant. The enzymes may be contained within the cell or cell free. Other enzymes
`
`capable of hydrolyzing hazardous chemicals such as OP compounds may be employed.
`
`Likewise, enzymes such as triesterase may be used for the decontamination of pesticides in a
`
`similar manner as herein described. Preferred enzymes are those that may be reactivated or
`
`directly hydrolyzed OP compounds.
`
`In another embodiment, the invention relates to the process of making a material, for
`
`the removal, decontamination or neutralization of hazardous chemicals such as OP
`
`compounds, comprising a mixture of enzymes immobilized on a porous support. In this
`
`embodiment, a mixture of enzymes and a prepolymer are gently and evenly mixed together
`
`with minimal degradation of the biotype component so that the resulting immobilized enzyme
`
`may efl’ectively decontaminate, neutralize or detoxify an amount of an OP compound. The
`
`device utilized, folds the components into one another. This is a low shear process. During
`
`synthesis of the material by prior art methods, for example a mixing drill, the enzymes utilized
`
`are subjected to fluid forces or shear stress. Use of a device that gently folds the components
`
`into one another greatly reduces these fluid forces or shear stress, and is the preferred device
`
`for enzymes, specifically enzymes that are sensitive to the high shear forces of the drill mixing
`
`device. Additionally, use of additives such as surface-acting polymers, e. g. P-65, or low
`
`concentrations of glycerol protects against enzyme denaturation induced by shear forces. The
`
`

`

`WO 00/64957
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`' 5 '
`
`PCT/USOO/11072
`
`surface-acting polymers also gives appropriate consistency and absorbency of the solid
`
`support.
`
`In a preferred process of making the material, a two chamber apparatus is utilized.
`
`One chamber contains a mixture of enzymes and the other chamber contains the prepolymer.
`
`The mixture of enzymes and the prepolymer are simultaneously extruded at a 1:1 ratio and
`
`mixed. Preferably, the mixture of enzymes and the prepolymer are rapidly and evenly extruded
`
`through a static mixing stator which gently and evenly mixes the enzymes and prepolymer. A
`
`preferred low shear device is a double chamber syringe and a static mixing stator typically
`
`used to mix viscous polyurethanes or epoxy glues. The size of the apparatus may vary
`
`depending on need, It may be pocketsize for use in the field by soldiers. Alternatively, the
`
`apparatus may be suitable for large—scale production and/or decontamination of a large objects
`
`or area. The low shear mixing device more than doubles the resultant AChE or BChE
`
`immobilized enzyme activity when compared to an identical mixture prepared with the high
`
`shear device.
`
`The invention further relates to various materials, methods and devices for reactivating
`
`the enzymatic activity of the material. These materials, methods and devices will allow a
`
`person to use the decontamination material of the invention for several separate uses and/or
`
`for a single and continuous use, which would normally require several decontamination
`
`materials but for reactivation of the enzymatic activity of the immobilized enzymes.
`
`Additionally, these materials, methods and devices allow for complete decontamination and/or
`
`neutralization of excess OP compounds absorbed by the porous support but did not react with
`
`the immobilized enzymes. These methods and reactivation materials employ substrates and/or
`
`oximes, to reactivate the catalytic activity of the OP inhibited and immobilized enzymes.
`
`The invention further relates to various materials and additives that are added to the
`
`embodiment to aid in the removal and decontamination of organophosphates from surfaces
`
`such as cracks, crevices, porous or uneven surfaces such as clothes and biological surfaces that
`
`readily absorb the organophosphates or pesticides such as skin. The additives are used in
`
`conjunction with the sponge material and may be incorporated within the porous support of
`
`the material. The additives may be in a dry or liquid form, and may be organophosphate
`
`solubilizing compounds such as triacetin or tetraglyme, or oximes, which both aid in
`
`decontaminating and reactivating enzymes.
`
`Another embodiment of the invention relates to a variety of kits. These kits contain
`
`the sponge containing a plurality of enzymes needed for the decontamination of
`
`

`

`wo 00/64957
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`PCT/USOO/11072
`
`organophosphorus and/or sulfiir compounds. Also included may be materials which would
`
`facilitate or be deemed necessary for the decontamination process. Kits may also include
`
`polymeric materials and enzymes if the foam is transient in nature, e. g. the prepolymer, a stable
`
`enzyme mixture and a low shear apparatus for making an organophosphorus and/or
`
`organosulfur decontamination foam. These kits may also include the indicators for both
`
`quantitative or qualitative detection of OP compounds and means for transmitting results to a
`
`central collection point, e.g. computer, satellite uplinks, radio relays, handheld battery
`
`operated measuring devices, etc. For example, one may quantitatively analyze the OP
`
`compounds by using a handheld battery operated measuring devices and interfacing with a
`
`computer to calculate reaction rates which rates may be relayed to a central collection point.
`
`The kits may contain items to facilitate the use of the device, e. g. instructions, containers, test
`
`tubes, etc.
`
`Description of the Drawings
`
`This invention is further understood by reference to the drawings wherein:
`
`Figure 1A illustrates the modeled surfaces of acetylcholinesterase, butyrycholinesterase
`
`and phosphotriesterase. Figure 1B illustrates the modeled surfaces of laccase.
`
`Figure 2 shows a cured material.
`
`Figure 3 schematically illustrates the specific reaction of the enzymes with prepolymer.
`
`Figure 4 shows the linear correlation between the amount of BChE added during
`
`synthesis of the material and the amount of BChE in the final material.
`
`Figure 5 shows the increasing amounts of BSA added during synthesis to a constant
`
`amount of AChE and TDI polymer.
`
`Figure 6 illustrates that the materials maintained enzymatic stability for more than 3
`
`years at 4°C and more than 12 months at 25°C and 45°C.
`
`Figure 7 shows that the material maintained enzymatic activity after consecutive
`washes.
`
`Figure 8 shows a substrate concentration dependent curve for soluble and polyurethane
`
`coupled AChE.
`
`Figure 9 illustrates the pH range of soluble and immobilized AChE.
`
`Figure 10 shows the relative activities of co-immobilized ChEs and OPHs.
`
`Figures 11A and B show a version of a manual mixing gun and a disposable mixing
`
`stator.
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`wo 00/154957
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`PCT/US00/11072
`
`Figure 12 schematically illustrates alternate schemes for detecting ChE activity.
`
`Figure 13 is a model of a carbon electrode with immobilized ChE.
`
`Figure 14 illustrates how F' reverses the reaction between an OP compound and ChE.
`
`Figure 15 illustrates how oximes may reactivate alkylphosphorylated ChE.
`
`Figure 16A illustrates the enzyme activity of immobilized FBS—AChE. Figure 16B
`
`illustrates the enzyme activity of immobilized Eq-BChE.
`
`Figure 17 represents inhibition of foam-immobilized FBS-AChE by DFP and
`
`reactivation by Ell-6.
`
`Figure 18 represents inhibition of foam-immobilized Eq-BChE by DFP and reactivation
`
`by TNIB4.
`
`Figure 19A shows about a 10-fold increase in Km because a shifi to the right is also
`
`observed in the immobilized (sponge) form when determined using the substrate paraoxon.
`
`On the other hand, Figure 19B shows shows little change in Km for the substrate paraoxon,
`
`with OPAA (derived from Alteromonas).
`
`Figure 20A shows the pH profile of soluble and immobilized choline oxidase. Figure
`
`20B shows substrate concentration dependent curve for soluble and polyurethane coupled
`choline oxidase.
`
`Figure 21A shows temperature profile of immobilized and soluble AChE. Figure 21B
`
`shows temperature profile of immobilized and soluble BChE.
`
`Figure 22 shows that only at very high ratios of organophosphate (lOOO—fold molar
`
`excess) is the process of binding, reactivation, and detoxification not complete. However,
`
`fresh HI—6 can restore most of the original activity once again.
`
`Figure 23 shows inhibition of AChE-sensor by the organophosphate MEPQ, which is
`
`not reversed by washing in water or buffer.
`
`Figure 24A shows protection afforded by sponge with tetraglyme additive. Figure 24B
`
`shows protection afforded by sponge with H1-6 additive. Figure 24C shows protection
`
`afforded by sponge with 2-PAM additive.
`
`Figure 25 illustrates the capacity of the resulting carbon sponge for binding methylene
`
`blue (a colorimetric indicator for activated carbon).
`
`Figure 26A shows AChE-sensor activities afier continuous incubation at 25°C at
`
`different pHs. Figure 26B shows BChE-sensor activities after continuous incubation at 25°C
`
`at different pHs. Figure 26C shows AChE-sensor activity after continuous exposure to
`
`Chesapeake Bay (Brackish) water at 25°C. Figure 26D shows AChE-sensor activity after
`
`

`

`wo 00/64957
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`' 8 '
`
`pcrmsoomon
`
`continuous exposure to Allegheny River (Fresh) water at 25°C. Figure 26E shows sensitivity
`
`of M272 ticket to aqueous conditions (Chesapeake Bay brackish water). Figure 26F shows
`
`sensitivity of M272 ticket to aqueoUs conditions (50 mM phosphate buffer, pH 8.0).
`
`Figure 27A shows dose-dependent inhibition of immobilized AChE sensor and soluble
`
`AChE to the pesticide dichlorophos. Figure 27B shows dose-dependent inhibition of
`
`immobilized AChE (sensor) and soluble AChE to the organophosphate soman (GD).
`
`Detailed Description of the Invention
`
`Enzymes have been incorporated in hypo-based urethane foam during polymer
`
`synthesis. See US. Patent No. 4,342,834. Hypoprepolymer is synthesized from a reaction of
`
`polyether (or polyester) polyol with isocyanates in the presence of cross-linking agents. See
`
`Havens, P.L., et £11., IndEng Chem Res (1993) 32:2254-2258; US. Patent No. 4,137,200;
`
`LeJeune, K.E., et al., Biotechnology and Bioengineering (1999) 20;62(6):659-665. Synthesis
`
`is initiated by bringing water molecules into contact with isocyanate groups present within the
`
`polyurethane prepolymer.
`
`A two—step procedure occurs from this point. Isocyanates react with water to form an
`
`unstable carbonic acid, which in turn degrades to an amine yielding C02 that gives the porous
`
`support lift and enables it to rise. The amines readily react with isocyanate groups, leading to
`
`production of urea type linkages. Since the enzyme contains multiple functional groups, such
`
`as amines and hydroxyls that can react with isocyanates, the enzyme becomes an integral part
`
`of the porous support during synthesis. Significant quantities of enzyme can link to the porous
`
`support without disrupting the progress of polymer synthesis. The reaction occurring during
`
`the polymer synthesis is shown below.
`
`1.
`
`C02 Evolution:
`
`co2
`
`i
`
`R1—NH2
`
`\OH
`
`o g
`
`R1—NCO + H20-——> R1—N’
`H
`
`2.
`
`Urea Linkage:
`
`R1—NH2
`
`+ Rz—NCO ——>
`
`3.
`
`Amine Group Enzyme Immobilization:
`
`E——-NH2 +
`
`R—NCO ——>-
`
`E
`
`c
`‘ ’ \ ’
`
`R
`
`

`

`wo 00/64957
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`PCT/USOO/11072
`
`4.
`
`Hydroxyl Group Enzyme Immobilization:
`
`E—OH + R—Nco—r»
`
`O
`
`E
`
`c
`\o’ \i-i/
`
`R
`
`The following list of enzymes and chemicals are examples of those suitable for use in
`
`the instant invention:
`
`Acetylcholinesterase (AChE);
`
`Butyrylcholinesterase (BChE);
`
`Pseudocholinesterase;
`
`Organophosphate hydrolases (OPH);
`
`Organophosphate acid anhydrase(OPAA);
`
`Phosphotriesterase;
`
`Pseudomonas diminuta bacterial OPH (paraoxonase);
`
`Laccases;
`
`Pralidoxirne chloride (2—PAM);
`
`7-(methoxyphosphinyloxy)— 1 -methy1quinolium iodide (MEPQ);
`
`Diisopropyl fluorophosphate (DFP);
`
`Acetylthiocholine iodide (ATC);
`
`S-butyrylthiocholine iodide (BTC);
`
`5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB);
`
`N,N'-trimethylene bis(pyridinium-4-aldoxime) dibromide (TMB4); and
`
`l-(2-hydroxyiminomethyl— l -pyridinium)-l -(4-carboxyaminopyridinium)-dimethy1ether
`
`hydrochloride (HI-6).
`
`By using mammalian cholinesterases such as FBS—AChE or Eq-BChE rather than Eel
`
`cholinesterase as is found in the M272 ticket (currently used to detect organophosphate
`
`compounds), the immobilized enzyme will display the same sensitivity to CPS that mankind is
`
`susceptible to now, or any new or novel OPS that might be produced in the future against
`
`mankind. Other enzymes capable of hydrolyzing hazardous chemicals such as OP compounds
`
`may be employed, for example laccase. Additionally, other OP hydrolyzing enzymes would
`
`ensure rapid and complete destruction of any toxic intermediates (for example,
`
`phosphoryloximes) that might be generated during the decontamination process.
`
`The following examples are intended to illustrate but not to limit the invention.
`
`

`

`wo 00/64957 ’
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`PCT/USOO/11072
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`E_xampl_e_1
`
`Determination of P0 ssible En_zyme Interference
`
`As polyether prepolymer derived from tolyl diisocyanate (TDI), reacts most favorably
`
`with free aliphatic amines such as lysine and arginine present on the surface of the ChEs (or
`
`any protein) to become a permanent cross-linked part of the material, computer aided
`
`molecular modeling of the enzymes was performed to highlight the available amino groups on
`
`the surface of each enzyme, and to determine whether the coupling of these groups to a
`
`porous support would interfere with enzymatic fianction. This may be performed on every
`
`enzyme for which its crystal structure is known, or enzymes which may be modeled by
`
`homology.
`
`Figure 1 A illustrates the modeled surfaces of acetylcholinesterase,
`
`butyrycholinesterase and phosphotriesterase and shows the lysine and arginine residues on the
`
`surface of the ChEs which are available for coupling to the prepolymer. This was generated
`
`by Insight 11, molecular modeling sofiware, by Biosym Technologies. Based on the molecular
`
`modeling, there are at least one lysine and 29 arginine water—accessible residues on the surface
`
`ofFBS-AChE to couple to the porous support, while 26 lysine and 26 arginine residues were
`
`modeled for equine-BChE. The majority of the lysine and arginine residues were found on the
`
`backside of the ChEs, and only a few are found on the side of the enzyme where the catalytic
`
`site gorge is located. The rim and the catalytic site gorge opening of both AChE and BChE
`
`appeared to be essentially devoid of lysine and arginine. Therefore, coupling these enzymes to
`
`the porous support should have minimal effect on the entrance of substrate, inhibitors such as
`
`OPS, or reactivators such as oximes which includes mono-bisquartemary oximes, release of
`
`products of catalysis to and from the active site, and the kinetic rates of the enzymes.
`
`Similarly, a model of the surface of laccase (Figure l B) is shown with available residues to
`
`couple covalently to the prepolymer.
`
`Example 2
`
`Synthesis of an Enzyme Bound Polflrethane Material
`
`A typical synthesis of the material comprises mixing enzymes in phosphate buffer
`
`containing 1% (final concentration) surfactant with prepolymer. Polyether prepolymer derived
`
`from tolyl diisocyanate (TDI), Hypol prepolymer TDI 3000 (Hampshire Chemical, Lexington,
`
`MA), and Pluronic P-65 surfactant (BASF Specialty Chemicals, Parsippany, NJ) were used.
`
`

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`wo 00/64957
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`PCT/USOO/11072
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`The 2-phase system is mixed and placed into a suitable mold and left to cure. Figure 2 shows
`
`a cured material which comprises a sponge-like support.
`
`Figure 3 schematically illustrates the specific reaction of the enzymes with prepolymer.
`
`Synthesis begins when H20 molecules react with the isocyanate groups present within the
`
`polyurethane prepolymer. Isocyanate reacts with the water to form an unstable carbonic acid,
`
`which degrades to an amine yielding C02. The C02 causes the polymer to rise and become
`
`porous, and simultaneously the amines readily react with the isocyanate groups leading to urea
`
`linkages.
`
`Since the ChE contains amines that are on the surface and available to react with the
`
`isocyanate groups, they can become an integral part of the polyurethane support during
`
`synthesis. There is no significant entrapment of the enzyme in the material as found with
`
`cyclodextrins, or physical adsorption of the enzymes, as observed with activated carbon. The
`
`inclusion of a surfactant such as Pluronic P-65 at about 1% final concentration controls the
`
`final structure and absorption potential of the material.
`
`To create a material comprising a porous polyurethane support, approximately 30 mL
`
`of 50 mM phosphate buffer, pH 8.0, containing P-65 surfactant buffer, was placed in a 600
`
`mL plastic beaker. 3 to 5 mL of either purified FBS-AChE (7500 units) or purified Eq-BChE
`
`(5000 units) was added, followed by approximately 40 gm of Hypo 3000 prepolymer (tolyl
`
`diisocyanate). The two-phase system was mixed and the material was allowed to expand for
`
`10 min, extruded from the container. The material was washed thoroughly with 50 mM
`
`phosphate buffer, pH 8.0, dried and stored in a zippered bag at 4°C for fiiture use.
`
`m3.
`
`Characteristics of Synthesized Material
`
`Approximately 20-90% of the enzymes were covalently linked to the porous support
`
`through free amino- or hydroxyl groups. This was determined by the presence of enzyme in
`
`first and second washes of the material.
`
`Since the enzymes can be attached at multiple points, they become a part of the cross-
`
`linked polymer support. The cross-linked polymer support imparts considerable stability to
`
`the bound enzymes. A large quantity of enzyme can be incorporated into a small polyurethane
`
`support, thereby rendering the cross-linked polymer support a highly effective material for
`
`decontamination .
`
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`PCT/US00/11072
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`A.
`
`Enzymatic Activity
`
`Five samples of materials containing PBS-AChE and five samples of materials
`
`containing Eq—BChE, ranging in weight from 1 to 40 mg, were suspended in 2.8 mL of 50 mM
`
`phosphate buffer, pH 8.0, and assayed using the method of Ellman. See Ellman, G.L., et al.,
`
`(1961) Biochem Pharmacol. 7:88-95. A linear correlation was found between the weight of
`
`the sponge and enzyme activity for both FBS-AChE and Eq-BChE immobilizations. See
`
`Figures 16A and B. The linear correlation between the weight of the material and enzyme
`
`activity indicates a uniform immobilization of AChE or BChE throughout the material.
`
`The material was washed with either 50 leI phosphate bufi‘er, distilled water, or 10
`
`mM ammonium bicarbonate without affecting substrate hydrolysis. Therefore, the mixing of
`
`prepolymer, surfactant, and enzyme in situ at 22°C yields a useful and effective material
`
`retaining about 50% of the original activity of soluble ChE.
`
`B.
`
`Protein Loading Capacity
`
`The material has a significantly higher loading capacity for ChEs such as BChE or
`
`AChE. The final activity of the BChE immobilized in the material could be increased by
`
`adding larger quantities of enzyme during synthesis. See Figure 4. When nonspecific protein
`
`(bovine serum albumin, BSA) was added to a constant amount of purified AChE, there was no
`
`reduction in ChE activity. See Figure 5. Thus, higher potency materials may be synthesized
`
`with additional proteins, enzymes and other ChEs. Additionally, materials effective against a
`
`diverse array of OP compounds may be readily synthesized by with combinations of multiple
`
`enzymes or a plurality of enzymes.
`
`C.
`
`Enzymatic Stability
`
`As illustrated by Figure 6, the immobilized ChE and OP hydrolase maintained
`
`enzymatic stability for more than 3 years at 4°C, and more than 12 months at 25°C and 45°C,
`
`respectively. If the material is frozen in liquid nitrogen, most of the original activity remains.
`
`TDI imparts remarkable stability to the immobilized ChE; about 50% of the original activity of
`
`the immobilized AChE and 20% of the activity of the immobilized BChE remained after 16
`
`hours at 80°C, conditions under which the soluble enzymes would exhibit no activity. The
`
`ChE materials can be exhaustively dried under vacuum at 22°C and then rehydrated without
`
`loss of enzyme activity. When AChE or BChE materials were exhaustively washed and
`
`assayed for activity, the wash and assay cycle repeated

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