WO2015168284A1

WO2015168284A1 - Albumin-bche in detoxification of neurotoxins

Info

Publication number: WO2015168284A1
Application number: PCT/US2015/028291
Authority: WO
Inventors: Merav Bassan; Aviva GROSS; Mallika Singh; Matthew M. SEAVEY; Jason Bock; Rom E. Eliaz
Original assignee: Teva Pharmaceutical Industries Ltd
Current assignee: Teva Pharmaceutical Industries Ltd
Priority date: 2014-04-30
Filing date: 2015-04-29
Publication date: 2015-11-05
Anticipated expiration: 2016-10-30

Abstract

The present invention provides the recombinant fusion protein of SEQ ID No :1. and a method of treating a mammal for exposure to a neurotoxin or for preventing or ameliorating the Neurotoxic Symptoms of exposure to neurotoxin which comprises administering an effective amount of the recombinant fusion protein of SEQ ID No :1. SEQ ID No:1 is a fully recombinant fusion polypeptide composed of the mature form of human serum albumin (HSA) fused at its amino terminus to the carboxy- terminus of a truncated and mutated human BChE.

Description

ALBUMIN-BCHE IN DETOXIFICATION OF NEUROTOXINS

This application claims the priority of U.S. Provisional Application No. 61/986,227, filed April 30, 2014, the contents of which are hereby incorporated by reference.

Throughout this application, various publications are referenced by author and publication date. Full citations for these publications may be found at the end of the specification immediately preceding the claims, The disclosures of these publications are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

Background of the Invention

Nerve agents (NA) are highly lethal chemicals, of which many belong to the organophosphorus (OP) compound group; they are among the most toxic substances identified. OP compounds were originally developed for use as insecticides, but due to their extreme toxicity were adopted as weapons of chemical warfare. Nerve agents act by irreversibly inhibiting the enzyme acetylcholinesterase (AChE) which is responsible for the breakdown of acetylcholine in the synapse. Current antidotes to OP exposure include pretreatment with pyridostigmine coupled with therapeutic administration (via auto-injector) of an anti-convulsant, an oxime and atropine. All current treatments carry significant side-effect risks. The pretreatment of subjects exposed to NAs with a human biologic is an attractive alternative, as protection lasting 1-2 weeks could be afforded to those in potentially contaminated environments; the same approach could be used by emergency medical workers responding to a contamination event.

In the last 2 decades both human butyrylchoiinesterase (hBChE) purified from either human plasma or from transgenic goat milk, and acetylcholinesterase (AChE) from various sources were studied extensively as stoichiometric antidotes (single turnover) against intoxications with multiple LD50 doses of OP-based nerve agents and/or pesticides. Studies of combined treatment by cholinesterases and axime reactivators suggest that these mixtures may constitute a pseudo-catalytic antidotal concept with turnovers >1 , Two sources of hAChE were examined as potential bioscavengers: a- recombinant hAChE expressed in a human cell line, and b, hAChE expressed in transgenic plant ceils , Despite the fact that hAChE reacts faster than hBChE with some OPs, the latter (obtained from human plasma) has the advantage of being already recorded as a safe protein administered in numerous cases in humans with no apparent toxic side effects and with long circulatory half- life of the "naked" administered enzyme. In several cases biological half-life of exogenously administered plasma derived hBChE in humans were reported at 10-12 days (3 J . Recombinant enzyme products of either hBChE or hAChE are required to be encapsulated to comply with requirements of extended plasma stability.

In order for hBChE (or any bioscavenger ) to qualify as drug candidate against a variety of OPs {i.e., a broad-spectrum antidote}, it must fulfill several requirements; It should react rapidly with all types of OP threat nerve agents (e.g., sarin, soman, cyclosarin, tabun, VX, Russian VX) in the circulation so as to prevent them from reaching AChE in physiologically important targets. The ideal prophylaxis {or post-exposure treatment) would be to reduce the concentration of the toxic dose of OPs to below 1/10 of its LD50 value within 0.5-1 min (approximately 1 blood circulation time); Bioscavengers should protect by a tolerable protein dose at least against 2xLD50 s of OPs, preferably (but not necessarily) without the need of post-exposure additional standard therapy (e.g., atropine, oxime reactivators, anti- cohvuiseants) ; The Bioscavenger should be effective not only as a prophylactic drug taut also as a post-exposure treatment with or without standard therapeutic regimen, whenever such therapeutic window exists (e.g., skin contamination, long-term low-level exposure) and should have long circulatory half-life so that a single dose is sufficient to protect against OP intoxication for 2-3 days, preferably i week. Additional requirements for such a bioscavenger is that it should be produced from a reliable and safe source, obtainable by a large-scale production process that will guarantee continuous supply at low cost and that it has shelf-life stability of at least 3 years when stored at room temperature (RT) as a Iyophilxzed powder and 5 years at 4°C.

To date., the need for an efficious, safe and commercially available, long acting and diverse bioscavenger for the treatment of exposure to several types of Neurotoxins still remains .

Summary of the Invention

The present invention provides a method of treating a mammal for exposure to a neurotoxin or for preventing or ameliorating the Neurotoxic Symptoms of exposure to a neurotoxin which comprises administering an effective amount of a recombinant fusion protein whose amino acid sequence is set forth as SBQ ID No:l. SSQ ID Noil is a fully recomb nant fusion polypeptide composed of the mature form of human serum albumin (HSA) fused at its amino terminus to the carboxy-termlnus of a truncated and mutated hunian BChE ,

Brief Description o£ the Figures

Figure 1. Hydrolytic activity of huBuChE (Baxter) vs. the recombinant fusion protein of SBQ ID NO: 1 against BtCh substrate

Both the recombinant fusion protein of SSQ ID NO:l and huBuChE display linear concentration/activity relationships for the hydrolysis of BtCh under the conditions used. When compared to huBuChE, the recombinant fusion protein of SEQ ID NO ; 1 possesses approximately nine times less hydrolytic activity against BtCh on a per milligram basis. The recombinant fusion protein of SEQ ID NO:l (126,000 g/mol) is approximately 1.5 times the molecular weight of huBuChE (85,000 g/mol}. Thus, the difference in hydrolytic activity between huBuChE and the recombinant fusion protein of SSQ I^'D NO; 1 is actually closer to six-fold, which is similar to the value that has been previously reported (Yang ft, Zhan C-G et al, 2010); this modest difference in activity may be due to the use of a buffer at a slightly lower pH {^'7.0 used here, 7.5 used in the previous study) .

Figure 2 , Non-linear regression fit of the recombinant fusion protein of SEQ ID NO : 1 hydrolysis of BtCh substrate as determined by isothermal titration calorimetry

2Ά. A single injection experiment was performed {MicroCal Manual) and a substrate inhibition model was used to analyze the data and generate the required ¾ and Ki {substrate inhibition constant) of BtCh with the recombinant fusion protein of SEQ ID NO:l. BtCh was present at a final concentration of 250 μΜ in the reaction well as this would allow for maximal velocity without causing substantial substrate inhibition. The concentration of enzyme that generated a linear change in absorbance at 412nm over a 5 minute measurement period was determined before each iteration of the experiment; generally, 150 to 200 ng of enzyme per reaction well was sufficient to meet this criterion. 2B. Agent <GA, GB, GD, GF, VX, VR, or VM at varying concentrations from 1 pM to I rn ) and substrate (at a fixed 250 μΜ final concentration) were simultaneously added to the recombinant fusion protein of SEQ ID O:l samples, and the absorbance was measured continuously for five minutes at 412ran. The resulting curves were fit using a nonlinear regression analysis to determine the rate of inhibition at each agent concentration, which was then plotted as described by Hart and O'Brien (Hart GJO et al, 1973} to determine the inhibition rate constants for each agent tested. Inhibition rate constants of the recombinant fusion protein of SEQ ID NO: 1 with several OP nerve agents, determined using the continuous method. Error bars indicate standard deviation of three independent replicates. Inset shows only the values for the recombinant fusion protein of SEQ ID NO:l with VX and VM. In all cases, the numerical values for each OP are shown above the corresponding bar.

Figure 3. In vi o PK (by residual activation of the recombinant fusion protein of SEQ ID NO: 1 } ex vivo in guinea pigs

Pharmacodynamic time-course of the recombinant fusion protein of ID NO:l in guinea pigs {n = 3 per dose) after i-.v. administration of A) 25 mg/kg or B) 50 mg/kg. Error bars show the standard deviation in the activity level at each time point. The concentration of the recombinant fusion protein of SEQ ID NO:l was determined by comparison to a standard curve generated using purified enzyme.

F gure 4. Summary of Experimental Results etailed Description_„ j J!ffl⁹?^⁰¹¹

SEQ ID NO: 2 has the following amino acid sequence:

EDDIIIATKNGKVRG NLTVFGGTVTAFLGl PYAQPPLGRLRFKKPQSL'TKWSDI NATKY.A NSCCQNIDQSFPGFHGSEMWN NTDLSSDCLyLNVWIPAPKPKKATVLIWIYGGGFQTGTSS LHVYDGKELARVERVIVVSMNYRVGALGFIALPGNPEAPGN GLFDQQLALQWVQ iilAAFG GNPKSVTLFGSSSGAASVSLHLLSPGSHSLFTRAILQSGSFNAP¾AVTSLYEARNRTLNLA LTGCSRSNETEIIKCLRNKDPQEILL EAFVVPYGTPLGVNFGPTVDGDFLTDMPDILLELG QFKKTQILVGVNKDEG WFLVGGAPGFSKD NS 11 R EFQEGLK1FFPGVSEFGKESΪLFH YTD VDDQRPENYREALGDVVGDYKFICPALSF KFSEWG NAFFYYFEHRSSKLPWPEWM GVMHGYEIEFVFGLPLERRDKYT AEEILS SIVKRWANFAKYGNPNBTQNNSTSWPVF ST BQKYLTLiJTESTRIMTKLRAQOCRFWTSFFPKVDAHKSEVAHRF DLGEBNFKALVLIAFAQ YLQQCPFEDHVKLVNEVTEFAKTCVADESASNCDKSLH LFGDKLCTVATLRETYGEMADCC AKQEPERNECFLQKKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRY AAFTBCCQAAD AACLLPKLDBLRDEG ASSAKQRLKCASLQ FGERAFKAWA VARLSQRFPKAEFAEVS LVTDLTKVHTECCHGDLLECADDRADLAKYICE QDSISSKL E CCEKPLLEKSHCIAEVE DEMPADLPSLAADFVES DVCKjSIYASAKDyFLGMFLYEYARRHP DYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDSF PLVEEPQNLIKQNCSLFEQLGEY FQNALLVRYTK VPQVSTPTLVEVSR^LGKVGSKCCKHPEA RMPCAEDYLSVVLNQLCVL HE TPVSDRV KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIK QTALVELV HKPKATKEQIiKAV DDFAAFVEKCC AOOKETCFABEGKKLVAASQAALGL (SEQ ∑D NO:l)

As used herein,, and unless stated otherwise, each of the following terms shall have the definition set forth below.

As used herein, "effective," as in an amount effective to achieve an end, means the quantity of a component that is sufficient to yield an indicated therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensura e with a reasonable benefit/risk ratio when used in the manner of this disclosure. For example, an amount effective to treat a mammal or a human patient exposed to a neurotoxin. The specific effective amount will vary with such factors as the age and gender of the maRmtai, the particular condition being treated, the physical condition of the mammal, the type of neurotoxin or nerve agent to which the mamma1 was exposed to and the nature of concurrent therapy (if any), including Oximes therapy, and the specific formulations employed.

As used herein, "ameliorating" a neurotoxic symptom or neurotoxic poisoning means slowing, stopping, inhibiting or reversing the progression of the neurotoxic symptom or neurotoxic poisoning, and/or lessening, alleviating or removing the neurotoxic symptom or the neurotoxic poisoning.

A "Neurotoxin" or "Meutoxins" also referred to as "nerve agents" are substances, which are generally prepared by chemical synthesis or extracted from natural sources (natural or artificial toxic substances} . Neurotoxins may cause deleterious or undesirable effects to a mammal if inhaled, absorbed, ingested, or otherwise encountered because of their high reactivity with and inhibition of chol inesterases . Exposure to "Neutoxins" or "nerve agents" could foe through inhalation, ingestion and/or dermal exposure- Examples of Neurotoxins include but are not limited to "organophosphorus compounds" (OPC) or Organophosphates (OP) , such as diisopropylfiuorophosphate (DFP) , CA (tabun) , GB(sam), GD (soman), GE (cyclosarin) , CV, y£, VGiamiton), VM, VR {RVX or Russian VX) , VS, VX, and combinations thereof.

"Organophosphorate pesticides" include but are not limited to acephate, azinphos-methyl, bensulide, cadusafos, chlorethoxyfos, chlorpyrifos , chlorpyrifos methyl, chlorthiophos, coumaphos, dialifior, diazinon, diehlorvos {DDVP} , dierotophos, distethoate, dioxathion, disuifoton, ethion, ethoprop, ethyl parathion, fenaraiphos, fenitrothion, fenthion, fonofos, isazophos methyl, isofenphos, malathion, methamidophos, methidathion, methyl parathion, meyinphos, monocrotophos, naled, oxydemeton methyl, phorate, phosalone, phosraet , phosphamidon, phostebupirim, plrimiphos methyl,. profenofos, propetamphos, suifotepp, sulprofos, teiaephos, terbufos, tetraehlorvinphos, tribufos (JDEF) , trich lorfori.

"Nerve agent poisoning" or "neurotoxic poisoning" relate to deleterious or undesirable effects to a mammal that are the result of exposure to a Neurotoxin or a nerve agent.

Neurotoxic symptoms can be "mild", "moderate" or "severe" in nature. "Mild" symptoms include but are not limited to bronchoconstriction, chest tightness, dim or blurred vision, conjunctival injection, fasciculations at site of exposure,, increased sweating at site of exposure, mild increase in bronchial secretions, miosis (pupillary constriction) with eye pain or headache or rhinorrhea and combinations thereof. "Moderate" symptoms include coughing, wheezing, fasciculations, generalized weakness, nausea, vomiting, diarrhea, generalized weakness, shortness of breath, dyspnea and combinations thereof. "Severe" symptoms include coma, seizures, flaccid paralysis, apnea, severe bronchorrhea and bronchospasm, generalized fasciculations, generalized secretions and death.

"Acetylcholinesterase inhibitors" are chemicals whose primary toxic effect is to block the normal breakdown of the neurotransmitter acetylcholine. Acetylcholinesterase inhibitors occupy and block the site where the neurotransmitter, acetylcholine, attaches to the enzyme acetylcholinesterase. With toxic doses, the result is that excessive levels of the acetylcholine build up in the synapses and neuromuscular junctions and glands.

"Oximes" restore AChE activity by hydrolytically cleaving the ^«OPC"s from the active site of inhibited AChE, AChE reactivators are mono- or bis-quaternary pyridinium. salts bearing in their molecule a functional oxime group able to split the bond between the OPC inhibitor and the enzyme, releasing free functional enzymes, which are therefore able to be once again physiologically active in the organism. Examples of oxiiiie include but are not limited to: pralidoxime chloride (2-ΡΆΜ) , mesylate (P2S),H16, toxogonin, MMB-4, HS-6 and TMB4 {A. BARELLI) .

A "bioscavenger molecule" is any molecule derived from a biological source such as recombinant DNA technology and/or being expressed from a biological organism, such as mammalian or bacterial cell culture {for example not being artificially synthesized like a peptide or small molecule) , which can scavenge or bind/block/remove another molecule from the immediate environment which otherwise would have posed a threat to the host organism. Unlike receptor-based scavengers found on the surface of cells or small molecule scavengers, a bio-scavenger is a large molecular entity derived from a biological source which can scavenge (remove and deactivate) toxic substances that otherwise would pose a threat to the immediate tissue environment

List of Abbreviations

As used herein, the following abbreviations have the meaning set out below throughout this disclosure:

PD Pharmacodynamic

PK Pharmacokinetic

TBU Teva Biopharmaceuticais USA

US United States

VJFI Water for Injection

NA Nerve agents

OP organophosphorus

AChE acetylcholinesterase

h^'BChS human butyrylcholinesterase

SC Subcutaneous ly

IV Intravenously

IK intramuscularly

The present invention provides a method of treating a mammal for exposure to a neurotoxin or for preventing or ameliorating the Neurotoxic Symptoms of exposure to neurotoxin which comprises administering an effective amount of the recombinant fusion protein whose amino acid sequence is set forth as SEQ ID No.:l. SEQ ID No : I is a fully recombinant fusion polypeptide composed of the mature form of human serum albumin (HSA) fused at its amino terminus to the carboxy-terminus of a truncated and mutated human BChE.

In an embodiment, the invention is a method for preventing or ameliorating the neurotoxic symptoms of exposure to neurotoxin in a mammal which comprises administering an effective amount of the recombinant fusion protein whose amino acid sequence is set forth as SEQ ID No:l.

In a further embodiment, the invention is a method for preventing or ameliorating neurotoxic poisoning in a mammal which comprises administering an effective amount of the recombinant fusion protein whose amino acid sequence is set forth as SEQ ID No: 1.

In an embodiment, the invention is a method for preventing or ameliorating the neurotoxic symptoms of exposure to neurotoxin or for preventing or ameliorating neurotoxic poisoning in a mammal wherein the neurotoxic poisoning or neurotoxic symptoms are expressed in the central, the peripheral, or the autonomic nervous systems, or in skeletal muscles.

The neurotoxic poisoning or neurotoxic symptoms could be associated with mild, moderate or severe neurotoxic symptoms or combinations thereof. The mild symptoms could be selected but are not limited to; bronchoeonstriction* chest tightness, dim or blurred vision, conjunctival injection, fascicuiations at site of exposure, increased sweating at site of exposure, mild increase in bronchial secretions, miosis (pupillary constriction} with eye pain or headache or rhinorrhea and combinations thereof. The moderate symptoms could be selected but are not limited to: symptoms selected from: coughing, wheezing, fasciculations, generalized weakness, nausea, vomiting, diarrhea, generalized weakness, shortness of breath, dyspnea and combinations thereof.

The severe symptoms could be selected but are not limited to: symptoms selected from; coma, seizures, flaccid paralysis, apnea, severe bronchorrhea and foroochospasm, generalized fasciculations, generalized secretions, death and combinations thereof .

In yet another embodiment, the recombinant fusion protein whose amino acid sequence is set forth as SEQ ID No:l is administrated prior to exposure to said neurotoxin.

In one embodiment of the invention the fusion protein whose amino acid sequence is set forth as SEQ ID Mo : i is administrated at least 12 hours prior to exposure to said neurotoxin or at. least 24 hours prior to exposure to said neurotoxin or at least 48 hours prior to exposure to said neurotoxin or at least 72 hours prior to exposure to said neurotoxin

In one embodiment of the invention the fusion protein whose amino acid sequence is set forth as SEQ ID No:l is administrated immediately after exposure to said neurotoxin,

In an embodiment, the invention is a method for preventing or ameliorating the neurotoxic symptoms of exposure to neurotoxin or for preventing or ameliorating neurotoxic poisoning in a mammal wherein the neurotoxin is a natural or artificial toxic substance. In an embodiment, the neurotoxin is an organophosphorate pesticide or an organophosphorous compound. In an embodiment, the organophosphorous compound is sarin (0- isopropyl-methylphosphonofluorida te, GB) , VX {ethyl-S-2- diisopropylaminoethyi-phosphano-thiolate) , MEPQ (7- (inethylethoxyphosphinylOKy) -l-methylquinolinium iodide} , soman {pinacolylmethyl-phosphonofluoridate, GD} , DFP

{di isopylfluorophosphate paraoxon), malathion, parathion, tabun (GA) , and cyclosarin <GF) , Russian. VK <VR) , paraoxon, Chlorpyrifos-oxon, VM or a combination thereof.

In one embodiment of the invention the exposure to the neurotoxin is through inhalation, ingestion, dermal exposure or a combination thereof.

In one embodiment of the invention the fusion protein whose amino acid sequence is set forth as SEQ ID No:! is administered subcutaneously (SC), intravenously (IV) or intramuscularly (IM) .

In an embodiment, the invention is a method for preventing or ameliorating the neurotoxic symptoms of exposure to neurotoxin or for preventing or ameliorating neurotoxic poisoning in a mammal which comprises co-administering an effective amount of the recombinant fusion protein whose amino acid sequence is set forth as SEQ ID Ho: 1 with an oxime, wherein said oxime reactivates said bioscavenger molecule. In an embodiment, the oxime is selected from the group consisting of 2-PAM, H16, toxogonin, MMB-4, HS-6 TMB4, and combinations thereof.

This invention will be better understood by reference to the Examples which follow, which are set forth to aid in an understanding of the subject matter but are not intended to, and should not be construed to, limit in any way the claims which follow thereafter.

Study Product

SEQ ID No.1 is a fully recombinant fusion polypeptide with a molecular mass of approximately 14 Q kDa, It is composed of the mature form of human serum albumin- (KSA) fused at its amino terminus to the carboxy-terminus of a truncated and mutated human BChE. Four mutations were made in the BChE amino acid sequence in order to increase cocaine hydrolytic activity. In addition, the last 45 amino acids of native human BChE molecule are responsible for tetramerization of the native molecule and these amino acids were removed from the recombinant enzyme molecule to facilitate expression of the enzyme in monomer form.

Example 1 : In Vivo Experiments

1.1. Hydrolytic Activity against Butyrylthiocholine {Figure 1)

The catalytic activity of the recombinant fusion protein of SEQ ID No : 1 against butyrylthiocholine needed to be established under the conditions utilized at the United States Army Medical Research Institute of Chemical Defense {USAMRICD} . These data were used to determine appropriate enzyme and substrate concentrations for use in subsequent assays .

The recombinant fusion protein of SEQ ID No;l was resuspended according to manufacturer instructions. The seal of the vial containing recombinant fusion protein whose amino acid sequence is set forth as SEQ ID Noil was flipped off and a needle was placed on a 3ml syringe. 1.0 ml of WFI was withdrawn into the syringe while ensuring that no air was trapped within the syringe when measuring the desired volume . The needle was inserted through the septum at an approximately a 90° angle while holding the plunger. Once the needle penetrated the septum, it was directed towards the side of the vial and the plunger was depressed slowly. Once all the WFI was dispensed into the vial the needle was slowly removed from the septum. The vial gently rolled between the palms of the hand for at least 6 minutes (fully dissolved) or was put on a mini-rotator (Glas-Col) with 20rpm for 20±4 minutes after it was initially rolled by hand for approximately 1 minute. The vial was not vigorously shaken and each vial was gently tapped for several seconds upon completion of reconstitution .

The resuspended recombinant fusion protein of SEQ ID Not! was diluted to concentrations ranging from 10 nM tD 35 nM (assuming a molecular weight of 126,000 g/mol) using 0.1 M potassium phosphate buffer at pH 7.0. Two microliters of each enzyme solution was added to iOOpL of 0.1 M potassium phosphate buffer at pH 7.0 in the wells of polystyrene 96-well flat bottom plates (iTisher Scientific) . After mixing, IOOpL of a solution containing 0.5 mM butyrylthiocholine (BtCh) and 1 mM dithionitrobensoic acid (DTNB) (Ellman et ai, 1961) in 0.1 M potassium phosphate buffer at pH 7.0 was added to each well and the change in absorbar.ce at 412 nm was measured using a Speetromax plate reader (Molecular Devices) over a period of five minutes; only the linear portion of each assay curve was used for data analysis. A similar experiment was performed in parallel using human plasma-derived butyrylcholinesterase (huSuChS; Baxter Biosciences) ,

1.2. Inhibition Rate Constants Determined Using a Continuous Measurement Method (Figure 2A, 2B, Table 1)

A continuously measured competition assay was used to determine the inhibition rate constants for both the recombinant fusion protein of SEQ ID NO: 2 and huBuChE for several OP nerve agents as an alternative, more reproducible method . In order to quantitatively compare the efficiency with which the recombinant fusion protein of SEQ ID MO:l sequesters nerve agents as compared to ability of huBuChE to bind the same compounds, a continuous measurement method was used. This method utilized the principle of competitive inhibition and the fact that all nerve agent inhibition of BuCnE is an inactivating covalent modification of the active site (Hart GJO et ai_r 1973) . For this approach to be used, the KM of the enzyme with BtCh. must be determined. Isothermal titration calorimetry was used to determine the KM value of the recombinant fusion protein of SEQ ID NO ; l with BtCh due to the fact that the KKH was too low to measure using the standard 96-vell plate assays described in earlier experiments. In brief, while this continuous method is not a direct measurement of the bimolecular association of OP nerve agents with an enzyme,- this assay more closely mimics the conditions under which OP nerve agent would compete with a native substrate in vivo to achieve inhibition of either acetylcholinesterase or butyryleholinesterase . Interestingly, while the absolute values of the inhibition rate constants determined by the continuous method are riot, identical to the bimolecular association rate constants determined using the discontinuous method, the relative inhibitory capacity of each OP for the recombinant fusion protein of SEQ ID NO:l remains nearly the same. Finally, in experiments conducted as part of another study, values determined from the continuous method using acetylcholinesterase with the same panel of OPs closely approximate previously reported values for the same parameters (Hart GJO et al_f 1973} .

TABLE 1. Recombinant Fusion Protein of SEQ ID NO:l rate constants versus Baxter human plasraa derived BuChE

1,3. Reactivation Capacity of Various Oxinnss with the recombinant fusion protein of SBQ ID MO:i (Table 2)

Small molecules such as oximes have the potential to regenerate active cholinesterase enzyme after exposure to 0? nerve agents (an otherwise irreversible covalent inhibitor) . The time required to reactivate fifty percent of an OP- inhibited enzyme population (tl/2) is an indication of the efficiency with which a specific reactivator acts on an enzyme inhibited by a specific OP nerve agent. The tl/2 for several commonly tested reactivators was determined for the recombinant fusion protein of SEQ ID NO: 1 after inhibition by a panel of OP nerve agents.

Equal volumes of the recombinant fusion protein of SSQ ID N0:1 and dilate OP nerve agent in the buffers described above in previous experiments v?ere incubated at room temperature for 10 minutes to achieve complete inhibition of hutyrylchoiinestarase activity. Excess nerve agent was removed by passing the samples through a small volume G25./150 size exclusion column (Centrisep, Princeton Separations, Inc., Adelphia, NJ) . Inhibited enzyme samples and positive control, uninhibited enzyme samples were diluted and incubated in the presence of 1 mM Oxime or buffer control, as appropriate. Periodically over the course of 2 hours, the capacity of the incubated samples and controls to hydrolyze BtCh was measured using the general method described in previous experiments . The activity of the uninhibited enzyme over the 2 hour time course was fixed as the maximum possible activity, and the percentage of reactivation for the inhibited sample was determined as the fraction of the maximum possible at each time point. The percent reactivation was plotted versus time, and was then fit linearly to determine the half-time for reactivation (tl/2) ,

TABLE 2. Half-times of reactivation (tl/2) for

Recombinant Fusion Protein of SEQ ID NO : 1 in the presence of nerve agents and oxime reactivators

Note that ^VND' = None Detected and ^¾-" = Not Tested

Example 2 : In Vivo Experiments

2.1. Pharmacodynamic Determination of the recombinant fusion protein of SEQ XD NO: 1 Circulatory Stability (Figure 3)

The circulatory stability of any bioscavenger candidate is an important factor for determining the usefulness of the candidate for the purposes of protecting against nerve agent intoxication, and indeed rnay define the concept's} of operation (ConOps) under which the enzyme can be useful. The experiments described here were performed in order to determine the time frame after administration of the recombinant fusion protein of SEQ ID NO: I that enzyme activity could be detected in the plasma of injected guinea pigs. This information was required to design appropriate protective efficacy experiments (see the following experimental section} .

Male Hartley guinea pigs {300 - 350 grams, Charles River Laboratories) were injected intramuscularly (i.m.) in the rear thigh or intravascularly fi.v, ) via a commercially implanted carotid catheter with either 25 mg/kg or 50 mg/kg of recombinant fusion protein of SEQ ID NO:l (n = 3 animals per dose and route) . Blood samples (~5D ui each} were collected via toe nail clip into llthium-heparin lined collection microcuvettes (Sarstedt, Germany}., processed immediately to plasma and stored at -80 °C. Timepoints collected for i . a . administered drug were 0, 1, 3, 16, 24, 48, 72, and 190 hours, Timepoints for i.v. administered drug were 0, 0,5, 1, 4, 20, 28, 44, 72, and 190 hours. Plasma samples were examined for the capacity to hydroiyze the substrate butyrylthiochoiine using the assay conditions described above. The total amount of the recombinant fusion protein of SEQ ID NOil was determined in each sample by extrapolation using an in vitro generated standard curve of the recombinant fusion protein of SEQ ID NO:i with the same substrate. The BtCh hydrolysis values derived from the 0 hour samples of guinea pig plasma were subtracted as background from each sample to account for the presence of endogenous BuChS in the guinea pig plasma.

The recombinant fusion protein of SEQ ID NO:l activity appears to reach a maximum at or before 30 minutes after i.v. administration. Butyrylchoiinesterase activity returns to baseline levels by 28 hours (25 mg/kg dose) or 72 hours (50 mg/kg dose) after administration, indicating that the protein is cleared quickly from the system. Preliminary analyses indicate an elimination half-time for the recombinant fusion protein of SEQ ID K0;1 from guinea pig plasma of -60 minutes. In results not shown, plasma samples collected from guinea pigs injected i.ra. with either 25 or 50 mg/kg of the recombinant fusion protein of SEQ ID NO:l were found to have no enhanced butyrylcholinesterase activity above baseline levels at any of the time points sampled, suggesting that little or no of the recombinant fusion protein of SEQ ID NO:l enters the blood of guinea pigs after i.m. administration of a dose as high as 50 mg/kg.

2.2. Protective Bffxcacy Testing in Guinea Pigs {Table 3)

The experiment conducted is an initial proof-of-concept in vivo test to determine if any protection against OP nerve agent intoxication can be provided via prophylactic administration of the recombinant fusion protein of SEQ ID NO: 1.

Male Hartley guinea pigs (300-350 grams, purchased with implanted carotid catheters from Charles River Laboratories^'! were injected with the recombinant fusion protein of SEQ ID NOtl at 50 mg/kg i.v. Animals were exposed sufocutaneousiy (s.c.) to 2 x LD5G of the OP nerve agents GA (tabun) , GB (sarin) , GD (soman) , GF (cyclosarin) , VX, or VR at 30 minutes after enzyme injection (n = 3 animals/agent, with the exception of experiments with VX, as detailed below) . OP compounds were diluted in saline such that injection volumes were 50 to 200 μΐ per animal, and s.c. injections were made into the left side of the animal below the rib cage. In an additional experiment, 3 guinea pigs were administered 250 mg/kg of the recombinant fusion protein of SEQ ID NO:l and subsequently exposed to 2 x LDSOs of VX; in this experiment, two additional animals were tested with VX after injection with 50 mg/kg. All exposed animals were scored for survival at 24 hours post-OP exposure,

TABLE 3. Survival rates for guinea pigs exposed to nerve agents for 30 mins after adminst ation of the recombinant fusion protein of SEQ ID NO:l

OP Agent Dose of OP {]xg/ g) 24 hour Survival

-

GA 240 1 / 3

GB 8? 3 / 3

GD 56 3 / 3

GF 108 3 / 3

VX 18 2 / 5

VR 22.6 3 / 3 X* 18 3 / 3

*These animals received an enzyme dose approximately 5- foXd higher than that administered to the other animals, followed by exposure to 2 x LD50 dose of VX.

Discussion

The results indicate that the modified human butyrylchoiinesterase moiety present in of the recombinant fusion protein of SEQ ID NQ:1 is capable of hydrolyzing the substrate butyrylthiocholine, albeit at a -6-foid slo¾rer rate than the wild-type human enzyme. This enzymatic activity is inhibited by a broad spectrum, of OP nerve agents (GA, G8, GD, GF, VX, VR, and VM) , and this inhibition occurs with different bimolecular rates and inhibition rates, depending on the OP in question. Both the discontinuous and continuous methods of assessing OP interaction with the recombinant fusion protein of SEQ ID NO:l indicated that the relative capacity of these OPs to inhibit the recombinant fusion protein of SEQ ID NO: 1 can be rank ordered. GF > GD > VR > GB > GA > VX ≥ VM.

The capacity of HI 6 and other oximes to reactivate the recombinant fusion protein of SEQ ID N0:l's butyrylchoiinesterase activity after inhibition by a panel of nerve agents indicates that none of the oximes tested are expected to afford broad spectrum reactivation, but that HI6 does reactivate GF- and VR-inhibited recombinant fusion protein of SEQ ID NO:l at a rate sufficiently fast to potentially allow pseudo-catalytic hydrolysis of these OP compounds in. vivo. The reactivation rates for the recombinant fusion protein of SSQ ID NO:l inhibited by other OPs are much longer, and as such do not afford much promise for in vivo reactivation even in non-acute situations (when pseudo- catalysis is not required, and one is only attempting to regenerate active enzyme to provide protection against a subsequent exposure), because the circulatory half-life of the oximes is much shorter than the half-time for reactivation. Thus, the oximes would be expected to degrade or otherwise be cleared from circulation before causing significant reactivation in vivo of the recombinant fusion protein of SSQ ID MO:l. The pharmacodynamic profile of the recombinant fusion protein of SEQ ID NO;l in guinea pig plasma after i,v. administration indicates that this protein is extremely short lived in circulation, with a half-life of roughly one hour. This short half-life is most likely the reason that no increase in butyrylchoiinesterase activity was detected after i,m. administration, as this route of protein administration generally results in lower Cmax values and reduced bioavailability as compared with i.v. administration. The recombinant fusion protein of SEQ ID NO:i appears to be suited for use as an immediate post-exposure intervention for OP poisoning; this ConOp requires both rapid bioavailability of an enzyme in circulation after administration, and sufficient circulatory stability of the enzyme to outlast the toxicokinetics of the OP.

Finally, the results from protective efficacy tests of the recombinant fusion protein of SEQ: ID NOil administered as a prophylactic against a broad spectrum of OF agents indicates that if present in the blood at sufficient quantities at the time of exposure, of the recombinant fusion protein of SEQ ID fJO:l can afford symptom-free protection against 2 x LD50s of GB, GD> GF, VX, and VR. The amount of enzyme necessary to provide protection against VX (and presumably GA) is higher than that provided by a 50 mg/kg dose of the recombinant fusion protein of SEQ ID NO : 1. In the case of GA, this may be a consequence of the fact that the LD50 of GA is low compared to that of other OP nerve agents (240 pg/fcg, or -1500 nmoles/kg, for 2 x LD50s) ; at 50 mg/kg of the recombinant fusion protein of SSQ ID NG:1, the animals receive only -400 nmoles of enzyme per kg. The results with VX, however, suggest that the situation is more complex than a simple measure of the stoichiometry of OP and enzyme. For VX, the dose of OF that constitutes 2 x LD50s is 67 nmoles/kg, so a dose of 50 mg/kg represents a nearly seven-fold molar excess of enzyme over agent. The failure of the recombinant fusion protein of SEQ ID NO: I to provide symptom-free protection against VX is most likely a consequence of the low binding affinity of VX for the recombinant fusion protein of SEQ ID NOi 1 (reflected in the bimolecular rates and inhibition rates shown in Figures 2A and 2B) . Interestingly, this explanation may apply for GA as well, as the inhibition rates for GA with the recombinant fusion protein of SEQ ID NOrl are also low as compared to values for other CPs, By analogy, it is expected that the recombinant fusion protein of SEQ ID NO:l at 50 mg/kg would also not provide complete protection against VM, although this compound was not tested in vivo. Regardless of the reason for the poor ability of the recombinant fusion protein of SEQ ID RO;l to protect against 2 x LD50 of VX at SO mg/kg, the results also indicate that administration of fivefold more protein does provide protection against the same dose of VX,

Claims

Claim 1. A method for preventing or ameliorating a neurotoxic symptom of exposure to a neurotoxin in a mammal which comprises administering to the mammal an effective amount of a recombinant fusion protein whose amino acid sequence is set forth as SEQ ID Ho:l.

Claim 2. The method of claim 1 wherein the neurotoxic symptom is expressed in the central nervous system, the peripheral nervous system, the autonomic nervous systems, skeletal muscle, or a combination thereof.

Claim 3. The method of claim 1 or claim 2 wherein the neurotoxic symptom is a mild, a moderate or a severe neurotoxic symptom or a combination thereof.

Claim 4. A method for preventing or ameliorating neurotoxic poisoning in a mammal which comprises administering to the mammal an effective amount of a recombinant fusion protein whose amino acid sequence is set forth as SEQ I^'D No:l.

Claim 5. The method of claim 4 wherein the neurotoxic poisoning is of the central nervous system, the peripheral nervous system, the autonomic nervous systems, skeletal muscle, or a combination thereof.

Claim 6, The method of claim 4 or claim 5 wherein the administration of the recombinant fusion protein prevents or ameliorates a mild, a moderate, or a severe neurotoxic symptom of the neurotoxic poisoning, or a combination thereof.

Claim 7, The method of claim 3 or claim 6 wherein the mild neurotoxic symptom is bronchoconstriction, chest tightness, dim or blurred vision, conjunctival injection, fasciculations at the site of exposure, increased sweating at the site of exposure, mild increase in bronchial secretions, miosis (pupillary constriction) with eye pain or headache or rhinorrhea, or a combination thereof.

Claim 8. The method of any one of claims 3, 6, or 7, wherein the moderate neurotoxic symptom is coughing, wheezing, fasciculations, generalized weakness, nausea, vomiting, diarrhea, generalised weakness,, shortness of breath, dyspnea, or a combination thereof.

Claim 9. The method of any one of claims 3 or 6-8 wherein the severe neurotoxic symptom is coma, seizures, flaccid paralysis, apnea, severe bronchorrhea and hronchospasm, generalized fasciculations, generalized secretions, death, or a combination thereof.

Claim 10. The method of any one of claims 1-9 wherein the recombinant fusion protein is administrated prior to exposure to said neurotoxin.

Claim 11. The method of claim 10 wherein the recombinant fusion protein is administrated at least 12 hours prior to exposure to said neurotoxin.

Claim 12. The method of claim 10 wherein the recombinant fusion protein is administrated at least 24 hours prior to exposure to said neurotoxin.

Claim 13. The method of claim 10 wherein the recombinant fusion protein is administrated at least 48 hours prior to exposure to said neurotoxin.

Claim 14. The method of claim 10 wherein the recombinant fusion protein is administrated at least 72 hours prior to exposure to said neurotoxin.

Claim 15. The method of any one of claims 1-14 wherein the recombinant fusion protein is administrated after exposure to said neurotoxin.

Claim 16. The method of claim 15 wherein the recombinant fusion protein is administered within six hours of exposure to said neurotoxin.

Claim 17. The method of claim 15 wherein the recombinant fusion protein is administered within one hour of exposure to said neurotoxin.

Claim 16. The method of claim 15 wherein the recombinant fusion protein is administered within 30 minutes of exposure to said neurotoxin.

Claim 19. The method of claim 15 wherein the recombinant fusion protein is administered within 10 minutes of exposure to said neurotoxin.

Claim 20. The method of claim 15 wherein the recombinant fusion protein is administered within 5 minutes of exposure to said neurotoxin.

Claim 21. The method of any one of claims 1-20 wherein the neurotoxin is a natural or an artificial toxic substance.

Claim 22. The method of claim 21 wherein the neurotoxin is an organophosphorous compound.

Claim 23. The method of claim 22 wherein the organophosphorous compound is selected from the group consisting of sarin (O- isopropyl -fflethylphosphonof luoridate, GB) , VX jethyl-S-2- di isopropylaminoethyl-phosphano-thioiate} , MSPQ (7-

(methylethoxyphosphinyloxy) -l-raethylquinolinium iodide) , soman {pinacolylmethyl-phosphonofluoridate, GD} , QETP

(diisopylfluorophosphate paraoxon) , malathion, parathion, tabun (GA) , and cyclosarin (GF) , Russian VX (VR) , paraoxon, Chlorpyrifoa-oxon, VM and combinations thereof.

Claim 24, The method of claim 22 wherein the neurotoxin is an organophosphorate pesticide .

Claim 25. The method of any one of claims 1-24 wherein the exposure to the neurotoxin is through inhalation, ingestion, dermal exposure or a combination thereof,

Claim 26, The method of any one of claims 1-25, wherein the recombinant fusion protein is administered subcutaneously.

Claim 27, The method of any one of claims 1-25, wherein the recombinant fusion protein is administered intravenously (IV} ,

Claim 28. The method of any one of claims 1-25, wherein the recombinant fusion protein is administered intramuscularly fXM) .

Claim 29. The method of any one of claims 1-28, which further comprises co-administration of an oxime, wherein said oxime reactivates said recombinant fusion protein.

Claim 30. The method of claim 29 wherein said oxime is selected from the group consisting of 2-PAKE, HIS, toxogonin, MMB-4, HS-6, TMB4, and combinations thereof.

Claim 31. The method of any one of claims 29-30, wherein the amount of the recombinant fusion protein and the amount of the oxime when taken together is more effective to treat the mammal than when each agent is administered alone.

Claim 32. The method of any one of claims 29-31, wherein the amount of the recombinant fusion protein in combination with the amount of oxime is less than is clinically effective when administered alone .

Claim 33, The method of any one of claims 29-32, wherein the amount of the oxime in combination with the amount of the recombinant fusion protein is less than is clinically effective when administered alone. Claim 35. The method of any one of claims 1-34, wherein the effective amount of the recombinant fusion protein is 5 mg/kg to 250 mg/kg.