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US20060194301A1 - Large-scale production of human serum butyrylcholinesterase as a bioscavenger - Google Patents

Large-scale production of human serum butyrylcholinesterase as a bioscavenger Download PDF

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US20060194301A1
US20060194301A1 US11/244,173 US24417305A US2006194301A1 US 20060194301 A1 US20060194301 A1 US 20060194301A1 US 24417305 A US24417305 A US 24417305A US 2006194301 A1 US2006194301 A1 US 2006194301A1
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column
hubche
buffer
preparation
product
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Bhupendra Doctor
Ashima Saxena
Wei Sun
Chunyuan Luo
Prasanthi Tipparaju
Irwin Koplovitz
David Lenz
Michelle Ross
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Priority to US11/733,246 priority patent/US7754461B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates to a method for the large-scale production of human serum butyrylcholinesterase (HuBChE) from Cohn Fraction IV-4 paste.
  • Human serum butyrylcholinesterase Human serum butyrylcholinesterase
  • organophosphorus compounds in the form of nerve agents and pesticides poses an ever increasing threat to military and civilian populations.
  • the serious medical challenges posed by chemical warfare agents to both the military and civilian health care systems became evident in the Iran-Iraq conflict and the 1995 Tokyo subway incident, respectively.
  • the acute toxicity of OPs is usually attributed to their irreversible inhibition of acetylcholinesterase (AChE).
  • acetylcholine concentration manifests at the cholinergic synapses of both the peripheral and central nervous systems, which precipitates a cholinergic crisis characterized by miosis, increased tracheobronchial and salivary secretions, bronchoconstriction, bradycardia, fasciculations, behavioral incapacitation, muscular weakness, and convulsions, ultimately culminating in death by respiratory failure.
  • Current antidotal regiments for OP poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and post-exposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride.
  • ChEs Among the enzymes examined as potent scavengers of highly toxic OP nerve agents, significant advances have been made using ChEs. Exogenous administration of plasma-derived ChEs such as AChE from fetal bovine serum (FBS) and BChE from human and equine serum (Eq), in both rodent and non-human primate models, has been successfully used as a safe and efficacious prophylactic treatment to prevent poisoning by OP compounds. See Doctor et al. (2001) “New approaches to Medical protection against chemical warfare nerve agents” CHEMICAL WARFARE AGENTS: TOXICITY AT LOW LEVELS, NYC, CRC Press, pp. 191-214.
  • FBS fetal bovine serum
  • Eq human and equine serum
  • ChEs as prophylactic agents have the advantage of being single pretreatment scavengers capable of protecting against multiple LD 50 's of a wide variety of potent OPs without the requirement of additional post-exposure therapy.
  • HuBChE has several advantages as an exogenously administered prophylactic for human use. See Ashani (2000) Drug Dev. Res. 50:298-308. First, it reacts rapidly with all highly toxic OPs, offering a broad range of protection for nerve agents including, soman, sarin, tabun, and VX. Studies in mice, rats, guinea pigs and rhesus monkeys clearly demonstrated that HuBChE could function as an antidote for all OP nerve agents. See Raveh et al. (1993) Biochem. Pharmacol. 45:2465-2474; Brandeis et al. (1993) Pharmacol. Biochem. Behav. 46:889-896; Allon etal.
  • HuBChE Extrapolation of data obtained from prophylaxis experiments with HuBChE in four species suggests that a dose of 200 mg of HuBChE as a prophylactic treatment can protect humans from exposure of up to 2 LD 50 of soman. Smaller doses of 50 mg of enzyme would be sufficient to provide protection against low-level exposure to nerve agents.
  • it also has potential use for first responders (civilians) reacting to intentional/accidental nerve gas release or pesticide overexposure.
  • HuBChE catalyzes the hydrolysis of cocaine and short-acting muscle relaxants succinylcholine and mivacurium, it could be an effective treatment for cocaine intoxication, as well as succinylcholine- and mivacurium-induced apnea.
  • HuBChE as a bioscavenger for human use was to obtain sufficient amounts of purified enzyme for conducting animal and clinical studies. Although a procedure for the purification of HuBChE from human plasma, which contains about 2 mg of enzyme per liter of plasma, was described, this source is not suitable for producing large quantities of HuBChE for clinical and commercial uses.
  • the present invention generally relates to the large-scale production of HuBChE preparations.
  • the present invention provides a method for obtaining an amount of a human butyrylcholinesterase preparation which comprises subjecting about 2 or more kilograms, preferably about 10 to 500 kilograms, more preferably about 100 to 500 kilograms, most preferably about 300 to 500 kilograms, of Cohn Fraction IV-4 paste to affinity chromatography followed by anion exchange chromatography.
  • the amount of Cohn Fraction IV-4 is about 80 kilograms or more, preferably about 80 kilograms.
  • the method comprises diluting the Cohn Fraction IV-4 paste by about ten-fold with water to obtain a suspension and then adjusting the suspension to a pH of about 4.5 to about 5.5, preferably about 4.7 to about 5.2, more preferably about 4.8 to about 5.0, most preferably about 4.9.
  • the method comprises centrifuging the suspension using a continuous flow centrifuge at about 7400 to about 7900 rpm, preferably about 7500 to about 7800 rpm, more preferably about 7600 to about 7700 rpm, most preferably about 7663 rpm at a flow rate of about 2 to about 6 kilograms per minute, preferably about 3 to about 5 kilograms per minute, more preferably about 4 kilograms per minute to obtain a supernatant.
  • the method comprises adjusting the supernatant to a pH of about 7.0 to about 9.0, preferably about 7.5 to about 8.5, more preferably about 8.0.
  • the method comprises filtering the supernatant with a 0.65 ⁇ m filter cartridge.
  • the affinity chromatography and the anion exchange chromatography are performed once.
  • the affinity chromatography is conducted using a procainamide column.
  • the anion exchange chromatography is conducted using a DEAE sepharose fast flow column.
  • the amount of the human butyrylcholinesterase preparation obtained is about 60% w/w of that present in Cohn Fraction IV-4 paste.
  • the human butyrylcholinesterase preparation is about 99% or more pure.
  • the human butyrylcholinesterase preparation in lyophilized form is storage stable at about ⁇ 20° C. to about 45° C. and is also stable in circulation upon storage at about ⁇ 20° C. for at least two years.
  • the butyrylcholinesterase in the human butyrylcholinesterase preparation exhibits a mean retention time of more than about 70 hours and an elimination half-life of more than about 35 hours in macaques.
  • the human butyrylcholinesterase preparation is non-toxic.
  • the human butyrylcholinesterase preparation is physiological, histopathological, or behavioral non-toxic to a subject when administered thereto.
  • the present invention provides a human butyrylcholinesterase preparation made by subjecting about 2 or more kilograms, preferably about 10 to 500 kilograms, more preferably about 100 to 500 kilograms, most preferably about 300 to 500 kilograms, of Cohn Fraction IV-4 paste to affinity chromatography followed by anion exchange chromatography.
  • the amount of Cohn Fraction IV-4 is about 80 kilograms or more, preferably about 80 kilograms.
  • the method comprises diluting the Cohn Fraction IV-4 paste by about ten-fold with water to obtain a suspension and then adjusting the suspension to a pH of about 4.5 to about 5.5, preferably about 4.7 to about 5.2, more preferably about 4.8 to about 5.0, most preferably about 4.9.
  • the method comprises centrifuging the suspension using a continuous flow centrifuge at about 7400 to about 7900 rpm, preferably about 7500 to about 7800 rpm, more preferably about 7600 to about 7700 rpm, most preferably about 7663 rpm at a flow rate of about 2 to about 6 kilograms per minute, preferably about 3 to about 5 kilograms per minute, more preferably about 4 kilograms per minute to obtain a supernatant.
  • the method comprises adjusting the supernatant to a pH of about 7.0 to about 9.0, preferably about 7.5 to about 8.5, more preferably about 8.0.
  • the method comprises filtering the supernatant with a 0.65 ⁇ m filter cartridge.
  • the affinity chromatography and the anion exchange chromatography are performed once.
  • the affinity chromatography is conducted using a procainamide column.
  • the anion exchange chromatography is conducted using a DEAE sepharose fast flow column.
  • the amount of the human butyrylcholinesterase preparation obtained is about 60% w/w of that present in Cohn Fraction IV-4 paste.
  • the human butyrylcholinesterase preparation is about 99% or more pure.
  • the human butyrylcholinesterase preparation in lyophilized form is storage stable at about ⁇ 20° C. to about 45° C. and is also stable in circulation upon storage at about ⁇ 20° C. for at least two years.
  • the butyrylcholinesterase in the human butyrylcholinesterase preparation exhibits a mean retention time of more than about 70 hours and an elimination half-life of more than about 35 hours in macaques.
  • the human butyrylcholinesterase preparation is non-toxic.
  • the human butyrylcholinesterase preparation is physiological, histopathological, or behavioral non-toxic to a subject when administered thereto.
  • the human butyrylcholinesterase preparation is packaged as a single dose in an autoinjector.
  • the present invention provides a pharmaceutical preparation comprising the human butyrylcholinesterase preparation described herein and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating, preventing, or inhibiting toxicity to an organophosphorus compound in a subject which comprises administering to the subject the human butyrylcholinesterase preparation made by subjecting about 2 or more kilograms, preferably about 10 to 500 kilograms, more preferably about 100 to 500 kilograms, most preferably about 300 to 500 kilograms, of Cohn Fraction IV-4 paste to affinity chromatography followed by anion exchange chromatography.
  • the amount of Cohn Fraction IV-4 is about 80 kilograms or more, preferably about 80 kilograms.
  • the method comprises diluting the Cohn Fraction IV-4 paste by about ten-fold with water to obtain a suspension and then adjusting the suspension to a pH of about 4.5 to about 5.5, preferably about 4.7 to about 5.2, more preferably about 4.8 to about 5.0, most preferably about 4.9.
  • the method comprises centrifuging the suspension using a continuous flow centrifuge at about 7400 to about 7900 rpm, preferably about 7500 to about 7800 rpm, more preferably about 7600 to about 7700 rpm, most preferably about 7663 rpm at a flow rate of about 2 to about 6 kilograms per minute, preferably about 3 to about 5 kilograms per minute, more preferably about 4 kilograms per minute to obtain a supernatant.
  • the method comprises adjusting the supernatant to a pH of about 7.0 to about 9.0, preferably about 7.5 to about 8.5, more preferably about 8.0.
  • the method comprises filtering the supernatant with a 0.65 ⁇ m filter cartridge.
  • the affinity chromatography and the anion exchange chromatography are performed once.
  • the affinity chromatography is conducted using a procainamide column.
  • the anion exchange chromatography is conducted using a DEAE sepharose fast flow column.
  • the amount of the human butyrylcholinesterase preparation obtained is about 60% w/w of that present in Cohn Fraction IV-4 paste.
  • the human butyrylcholinesterase preparation is about 99% or more pure.
  • the human butyrylcholinesterase preparation in lyophilized form is storage stable at about ⁇ 20° C. to about 45° C. and is also stable in circulation upon storage at about ⁇ 20° C. for at least two years.
  • the butyrylcholinesterase in the human butyrylcholinesterase preparation exhibits a mean retention time of more than about 70 hours and an elimination half-life of more than about 35 hours in macaques.
  • the human butyrylcholinesterase preparation is non-toxic.
  • the human butyrylcholinesterase preparation is physiologically, histopathologically, or behaviorally non-toxic to a subject when administered thereto.
  • the human butyrylcholinesterase preparation is packaged as a single dose in an autoinjector.
  • the present invention provides a method of treating, preventing, or inhibiting toxicity to an organophosphorus compound in a subject which comprises administering to the subject a pharmaceutical preparation according to the present invention.
  • FIG. 1A is a schematic describing a method for obtaining Cohn Fraction IV-4.
  • FIG. 1B is a schematic describing the method according to the present invention for obtaining HuBChE from Cohn Fraction IV-4.
  • FIG. 2 is a graph depicting the elution of HuBChE from a procainamide affinity column Run # 1 .
  • the activity and A 280 of each fraction are shown.
  • the volume of each fraction (#4-32) was 800 ml and #33-35 was 20 L.
  • fractions 4 to 35 were pooled.
  • the pool (87 L) contained 8.4 million units of BChE activity.
  • FIG. 3 is a graph depicting the elution of HuBChE from a DEAE fast flow column Run # 1 .
  • the activity and A 280 of each fraction are shown.
  • the volume of each fraction was 500 ml.
  • fractions 3 to 26 were pooled.
  • the pool contained 5.5 million units of BChE activity.
  • FIG. 4 is a photograph of a 10% SDS-PAGE of HuBChE purified from Cohn Fraction IV-4.
  • FIG. 5 is a graph depicting the thermal stability of HuBChE stored at various temperatures, in lyophilized form. Aliquots of enzyme (1 mg) were stored in lyophilized form at 4° C. ( ⁇ ), 25° C. ( ⁇ ), 37° C. ( ⁇ ) or 45° C. ( ⁇ ).
  • FIG. 6 is a graph of the procainamide affinity column Run # 2 wherein activity and A 280 of each fraction are shown. A total of 37 fractions (#1 to #37), totaling about 53.2 kg of HuBChE were pooled.
  • FIG. 7 is a graph of the DEAE column Run # 2 wherein activity and A 280 of each fraction are shown. A total of 16 fractions (#7 to #22), totaling 16.75 kg of HuBChE were pooled.
  • Cohn Fraction IV-4 paste a by-product of human plasma generated during the production of human proteins, such as ⁇ -globulin, was identified as a rich source of HuBChE.
  • This paste contains about 150 mg of enzyme per kg, which is much higher than human plasma and contains much lesser quantities of other plasma proteins due to the fractionation steps deployed in the production process.
  • Cohn Fraction IV-4 is regarded as a waste product of the fractionated plasma and is disposed of after addition of large quantities of Celite®. This results in the inactivation of most of the enzyme activity reducing its activity to about 3 mg/kg, thereby rendering this paste unsuitable for the isolation of HuBChE. Therefore, the most suitable source of HuBChE is freshly prepared Cohn Fraction IV-4 paste prior to the addition of Celite®, a filter aid.
  • the enzyme did not exhibit any toxicity in mice or guinea pigs even at doses up to 30 and 60 mg/kg, respectively, as measured by general observation, serum chemistry, complete blood count, and gross and histological tissues changes.
  • HuBChE as prophylactic agent has the advantage of being a single pretreatment scavenger capable of protecting against multiple LD 50 's of a wide variety of potent OPs without the requirement of additional post-exposure therapy.
  • HuBChE will be the first successful pretreatment/treatment that can afford protection against not only mortality, but also against the adverse physiological and behavioral effects of nerve agent exposure. This technology will provide sustained maximum protection to the war fighter under the most adverse battlefield conditions without performance degradation or interruption of the pace of operations.
  • the invention pertains to a process as herein described, wherein the Cohn Fraction IV-4 paste is fresh.
  • the invention pertains to a process as herein described, wherein the Cohn Fraction IV-4 paste has not been subjected to an addition of Celite®.
  • the invention pertains to a process as herein described, wherein said resuspending comprises resuspending the Cohn Fraction IV-4 paste in water.
  • the water is CWFI.
  • the Cohn Fraction IV-4 paste can be resuspended in other pharmaceutically acceptable solvents.
  • the invention pertains to a process as herein described, wherein said adjusting comprises adjusting the pH of the solution to a pH about pH 8.0, prior to subjecting the solution to procainamide affinity chromatography.
  • the solution is adjusted to about pH 8.0 by treating the solution with a sodium hydroxide solution.
  • the invention pertains to a process as herein described, wherein said subjecting the solution to procainamide affinity chromatography comprises eluting the HuBChE from a chromatography column packed with procainamide sepharose gel using sodium chloride solution.
  • the invention pertains to a process as herein described, wherein said subjecting the solution to DEAE ion-exchange chromatography comprises eluting the HuBChE from a chromatography column packed with DEAE sepharose fast flow gel.
  • FIG. 1A and FIG. 1B An overview of the procedure that can be used to practice the present invention starting with plasma is shown in FIG. 1A and FIG. 1B .
  • Cohn Fraction IV-4 paste can be obtained frozen. However, as noted above, it is preferred that the Cohn Fraction IV-4 paste is fresh.
  • Step 1 A total of 80 kg of Cohn Fraction IV-4 paste (Medimmune Inc., MD) was resuspended in 720 L of Cold Water for Injection (CWFI). The resuspension was mixed for 6 to 8 hours at 0° C. to 4° C. The pH of the resuspension was adjusted to 4.90 (from 5.73) by the addition of 1.91 kg of 80 X concentrated sodium acetate (NaAc) buffer. The Fraction IV-4 resuspension was centrifuged in the Carr P 18 Powerfuge at 7,500 rpm at a feed flow rate of 4.0 kg/min. Temperature was maintained at 2° C. to 8° C. This resulted in 712 L of supernatant and 16.8 kg of paste.
  • CWFI Cold Water for Injection
  • Step 2 Three Cuno® 60LA filter cartridges were installed in a Cuno® filter housing and flushed with 100 L of water. A total of 0.75 kg of acid washed Celite® 521 was added to about 498 L of CWFI and mixed for 30 minutes. This mixture was then transferred through the Cuno® filters to precoat the filter surface. This was followed by a 200 L rinse with 20 mM NaAc buffer, pH 4.9. A total of 2.85 kg of acid washed Celite® 521 was added to the 712 L of product and mixed for about 1 hour (4 g filter aid/kg of product). The product was then filtered through the Cuno® filters.
  • the final filtered volume (including buffer chase) was about 963 L.
  • Step 3 The product was adjusted to pH 8.04 by the addition of 50.34 L of 0.4 N NaOH solution. This resulted in 1016 L of product. The product was then filtered through a 30 inch 0.65 ⁇ m filter cartridge. Final quantity after filtration was about 1005 L.
  • the procainamide sepharose column was equilibrated with a minimum of 5 column volumes of equilibration buffer (25 mM sodium phosphate pH 8.0 and 1 mM EDTA).
  • Product was loaded onto the column at about 1100 ml/min with a total loading time of about 16 hours.
  • the column was washed with 134 L equilibration buffer at 1100 ml/min.
  • the column was then washed with 1440 L equilibration buffer +0.075 M NaCl at about 155 ml/min.
  • Step 5 A Millipore® Pellicon apparatus, with 1.0 m 2 of Millipore® 100 kDa membrane, was washed with WFI and equilibration buffer prior to use.
  • the procainamide pool was concentrated to 9.8 L and then diafiltered with more than 16X volumes of equilibration buffer. The final diafiltered volume was about 12 L.
  • the DEAE column was equilibrated with a minimum of 5 column volumes of equilibration buffer. Diafiltered product was then loaded onto the column at a flow rate of 75 ml/min. Following loading, the column was washed with 58 L of equilibration buffer at 75 ml/min. The column was then washed with equilibration buffer +0.05 M NaCl at 75 ml/min for about 40 column volumes.
  • the column was eluted with a 10 column volume gradient from equilibration buffer +0.05 M NaCl (low) to equilibration buffer +0.3 M NaCl (high) at a flow rate of 75 ml/min. Fractions of 500 ml were collected manually starting at the first appearance of protein as detected by a flow through UV monitor A 280 . A total of about 25 L of product was pooled for further processing.
  • Step 7 A Millipore® Pellicon apparatus, assembled with 1.0 m 2 of Millipore® 100 kD membrane, was washed with WFI and equilibration buffer prior to use.
  • the DEAE pool was concentrated to 5 L and then diafiltered with equilibration buffer.
  • the final volume of the concentrated diafiltered product was about 8 L and about 5 million units corresponding to about 8 g of HuBChE as shown in FIG. 3 .
  • Step 8 A Millipore® Pellicon apparatus, assembled with 2.5 m 2 of Millipore® 30 kD membrane, was washed with WFI and equilibration buffer prior to use.
  • the DEAE pool from step 7 was concentrated further to obtain about 850 ml and about 5 million units corresponding to about 7.4 g of HuBChE.
  • the enzyme was filter sterilized using a 0.22 ⁇ m filter and 3 ml of solution containing 25 mg of HuBChE was aliquotted into 10 ml glass vials, lyophilized and stored at ⁇ 20° C.
  • BChE activity was determined spectrophotometrically at 25° C. according to the Ellman method. See Ellman et al. (1961) Biochem. Pharmacol. 7:88-95, which is herein incorporated by reference.
  • the assay mixture contained 1 mM butyrylthiocholine as the substrate and 1 mM 5,5-dithiobisnitrobenzoic acid in 50 mM sodium phosphate, pH 8.0.
  • One unit of the enzyme activity is defined as the amount required to hydrolyze 1 ⁇ mol of substrate/min.
  • BChE protein was assayed by monitoring the absorbance at 280 nm. An extinction coefficient of 1.92 for a solution containing 1 mg/ml of HuBChE was used for calculating the specific activity of these preparations. Table 1 below shows the recovery of HuBChE activity and protein from Cohn Fraction IV-4 paste.
  • a dedicated viral elimination step may be included in this purification scheme after step 5 as follows: Calculate the amount of TNBP required to treat the product (0.003 ml/g). Calculate the amount of 20% Triton X-100 required to treat the product (0.0526 ml/g). Combine the TNBP and 20% triton X-100 and mix for 15 minutes. Slowly add the TNBP/Triton mixture to the product. Once all the solvent detergent has been added, mix for 15 minutes. Transfer the treated product to a clean container and hold for a minimum of 4 hours. Assay sample for total protein and BChE activity and load on the DEAE fast flow column.
  • Electrophoresis was performed on a 10% polyacrylamide gel according to the procedure of Laemmli known in the art. The reduced form was obtained by boiling enzyme samples with 5% mercaptoethanol for 5 minutes. Each lane was loaded with about 10 to about 20 ⁇ l sample containing about 5 to about 30 ⁇ g of protein. Protein bands were visualized by staining with Coomassie blue.
  • the samples were analyzed on a YMC-Pack-300, S-5 um, 30 nm column (300 ⁇ 6 mm ID) using 100 mM sodium phosphate, pH 8.0 +0.2 M NaCl as the liquid phase.
  • the column was calibrated using gel filtration standards from Bio-Rad; thyroglobulin (7.2 min), gamma globulin (8.9 min), ovalbumin (9.67 min), myoglobin (10.46 min), and vitamin B 12 (11.41 min).
  • the purity of the HuBChE preparations can be followed by specific activity, evidence of a single protein band on SDS-PAGE and HPLC.
  • the specific activity of the purified enzyme was about 700 U/mg measured in 50 mM sodium phosphate buffer at pH 8.0 at 25° C., using 1 mM butyrylthiocholine as the substrate.
  • Active-site titration of purified enzyme with 7-(O,O-diethylphosphinyloxy)-1-methylquinolinium methyl sulfate (DEPQ) revealed that 1 mg of enzyme contains 11 nmoles of active-sites. Each nmole of enzyme contains 64 to 68 U of activity.
  • the enzyme migrates as a single band on a reducing SDS-PAGE, with a subunit molecular weight of 85 kD as shown in FIG. 4 .
  • the size of the band is reduced to 65 kD upon treatment with N-glycosidase F.
  • the intact protein migrates as a single band on native polyacrylamide gel electrophoresis, which can be stained for enzyme activity.
  • the intact protein also migrates as a single peak on sucrose density gradients, with a sedimentation coefficient (S 20,w ) of 12.44, corresponding to the tetrameric form of the enzyme.
  • the enzyme elutes as a single peak at 7.42 minutes from a YMC-Pack-300 column.
  • the present invention provides an HuBChE preparation having 11 nmoles of active-sites per 1 mg and about 64 to about 68 U of activity per 1 nmole of enzyme.
  • the stability of the HuBChE preparations at different storage temperatures was examined. Aliquots of the HuBChE preparations in lyophilized (1 mg) or liquid form (10 mg/ml in 50 mM sodium phosphate, pH 8.0 +10% glycerol +1 mM EDTA) were stored at 4° C., 25° C., 37° C. or 45° C., and the enzyme activity. Samples were resuspended in 1 ml of 50 mM sodium phosphate buffer, pH 8.0 at various time intervals and assayed for BChE activity using the Ellman assay known in the art. The HuBChE preparations were found to be stable when stored in lyophilized from at 4° C., 25° C., 37° C. or 45° C. for over two years as shown in FIG. 5 .
  • the in vivo circulatory stability of the BChE of the HuBChE preparations was determined by measuring the pharmacokinetic profile of the enzyme (stored as lyophilized HuBChE preparations at ⁇ 20° C. for various time periods) following i.m. administration into CD-1 mice as described below.
  • the pharmacokinetic properties of the enzyme were not affected upon storage at ⁇ 20° C. for at least 2 years. In other words, the pharmacokinetic properties of the HuBChE preparations do not show observable change when stored at ⁇ 20° C. for about 2 or more years.
  • the HuBChE preparations of the present invention are storage stable.
  • the HuBChE preparations (60 mg/kg HuBChE) were administered to guinea pigs by a single i.p. or i.m. injection. Blood samples were taken for up to 14 days post-injection for the measurement of blood BChE activity.
  • the HuBChE in the HuBChE preparations exhibited a marked prolonged MRT of about 110 hours in guinea pigs, which was at least twice as long as in mice. Guinea pigs still displayed 8 to 12 U/ml of HuBChE activity in blood at 14 days post administration.
  • the HuBChE preparations (30 HuBChE mg/kg) were administered in four rhesus monkeys by an i.v. bolus injection. At 5 minutes post-injection, the mean level of BChE in the blood was 226 U/ml. The mean residence time was about 72 hours. The HuBChE activity returned to baseline levels at 2 weeks post injection.
  • HuBChE preparations 70 to 2100 HuBChE U/mouse were administered into CD-1 mice by a single i.p. or i.m. injection, as described above.
  • two weeks post-injection all animals were euthanized and blood was collected for the analysis of multiple serum chemistry parameters (including glucose, urea nitrogen, creatinin, Na, K, Cl, CO 2 , Ca, PHOS, cholesterol, total protein, triglycerides, albumin, AST, ALT, LDH, CK, ALKP, GGT, and total bili) and hematology parameters (including WBC, RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV). Tissues were removed for complete gross/histopathological examination to determine any potential toxicity in vital organ and system functions. No toxic side effects were suggested from any of these observed parameters.
  • the HuBChE preparations (60 HuBChE mg/kg) were administered to guinea pigs by a single i.p. or i.m. injection, as described above. Fourteen days post HuBChE injection, 20 panel serum chemistry, hematology, and complete gross/histopathology were examined in all animals. No toxic effects were found from these observed parameters.
  • the HuBChE preparations (about 30 HuBChE mg/kg) were given to each of four Rhesus monkeys by an i. v. bolus injection to determine the behavioral and physiological safety of the preparations using a complex cognitive test (Serial-Probe Recognition, SPR) designed to assess attention and short-term memory known in the art.
  • SPR Serial-Probe Recognition
  • Blood samples were taken at various time intervals for 4 weeks post-injection for the measurement of blood BChE activity, anti-BChE (antibody) production, serum chemistry, and hematology tests. No cognitive-behavioral decrements of any kind were detected in SPR performance tests and no robust or consistent signs of clinical pathology were detected in any of the blood assays during the 5 weeks of observation.
  • blood samples were withdrawn for the determination of BChE concentration.
  • the total number of moles of HuBChE available in circulation was calculated by assuming a total blood volume of 20 ml for a 400 g guinea pig.
  • Agent doses were about 1.5, 2.0 and 2.0 ⁇ LD 50 administered s.c.
  • the HuBChE preparations of the present invention may be used as a prophylactic treatment which may be advantageously administered as a single pretreatment capable of protecting against multiple LD 50 's of a wide variety of potent OP agents without the requirement of additional post-exposure therapy.
  • the HuBChE preparations according to the present invention can be packaged as storage stable single dose. The single doses may be packaged as prefilled syringes or autoinjectors.
  • the HuBChE preparations according to the present invention may be used to hydrolyze cocaine, succinylcholine, mivacurium, albuterol, and the like. Therefore, the HuBChE preparations of the present invention can be used for alleviating toxicity due to overdosing of these drugs.
  • the present invention provides a process for the large-scale manufacture of human butyrylcholinesterase (HuBChE) from Cohn Fraction IV-4 paste.
  • Three small-scale purifications were performed to evaluate the proposed manufacturing process. Following this, two large-scale purification runs were performed using 80 kg of Fraction IV-4 paste. A total of 9.4 million units of HuBChE were produced with a purity of about 99%, for the two runs. Both the yield and purity met or exceeded expectations for pre-clinical material. Based on the successful results of this study, a manufacturing procedure has been developed that is suitable for the pre-clinical production of HuBChE.
  • a total of 800 g of Cohn Fraction IV-4 paste (PN 527BE, Batch 02ZB005) was resuspended in 7200 g of 25 mM sodium phosphate, 1 mM EDTA, pH 8.0 (EQ Buffer) at room temperature. The resuspension was mixed for 17.5 hours at 2° C. to 8° C.
  • a Cuno® Biocap 1000 60LA filter capsule was flushed with 3 L of water. Acid washed Celite® 521 (5 g) was added to 5.5 L of EQ Buffer and flushed through the capsule to precoat the filter. Acid washed Celite® 521 was added to the product at a rate of 20 g/kg of resuspension (160 g total) and mixed for 30 minutes. Filtration was started at 110 ml/min. The flow rates rapidly declined necessitating filter replacement. Although a total of three filters were used, the entire product volume could not be filtered. The final filtered volume was about 10 L (including EQ buffer flushes). About 2 L of unfiltered product was discarded.
  • the Cuno® filtrate was filtered through one 1.2 ⁇ m Pall HDC II cartridge. It was then filtered through two 47 mm 0.65 ⁇ m Millipore® Durapore filter discs.
  • the packing flow rate was about 20 ml/min (about 1 cm/min linear flow rate).
  • the column was pre-cycled with EQ Buffer (about 10 CV), 2.0 M NaCl (about 3 CV), DI water (about 1.2 CV), 0.5 M HAc (about 1.4 CV), DI water (about 1 CV), and EQ Buffer (about 4 CV).
  • Flow rates ranged from I ml/min to 7 ml/min determined by convenience. Filtered product was loaded onto the column at flow rates between 6.5 ml/min and 7.7 ml/min. Overall loading time was 24 hours 40 minutes.
  • a Millipore® stirred cell diafiltration apparatus was assembled with a Millipore® 100 kD (YM100) membrane. The product was diafiltered with about 5X volumes of water and concentrated to a final volume of 125 ml.
  • the pH of the diafiltered pool was adjusted from 8.05 to 4.94 by the addition of 2.5 ml of 1 M NaAc pH 4.09. The pH was then adjusted to 4.02 by the addition of about 0.4 ml of HAc.
  • the product was filtered through a 1.2 ⁇ m membrane filter.
  • S/D was prepared by mixing 5.81 g of 20% Triton X-100 with 0.33 g of TNBP. The S/D was added to the filtered product and mixed overnight at 2° C. to 8° C.
  • the column was then washed with 20 mM NaAc pH 4.0 buffer +0.05 M NaCI at 4.0 ml/min for about 18 CVs. Elution was performed with EQ Buffer +0.2 M NaCi at a flow rate of 3.7 ml/min. Fractions (about 7.4 ml/fraction) were collected using a fraction collector. A total of 59 ml of product was pooled. The column was cleaned by use of 1.5 CVs of 2.0 M NaCl and stored in 20% (v/v) ethanol.
  • a Millipore stirred cell diafiltration apparatus was assembled with a Millipore 100 kD membrane. The product was concentrated to 25 ml and then diafiltered with about 5X volumes of EQ Buffer. Product was concentrated to a final volume of 15 ml.
  • a total of 800 g of Cohn Fraction IV-4 paste (PN 527BE, Batch 02ZB005) was resuspended in 7200 g of water at room temperature. The resuspension was mixed for about 7 hours at 2° C. to 8° C.
  • the pH of the resuspension was adjusted to 4.90 by the addition of 80X NaAc.
  • the resuspension was centrifuged using a Sorvall® RC5B refrigerated centrifuge with a GS3 rotor at 9000 rpm (15000 X g) for 8 minutes.
  • the final supernatant volume was 7750 ml.
  • a Cuno® Biocap 1000 60LA filter capsule was flushed with 3 L of water followed by 10 L of 20 mM NaAc, pH 4.9. Acid washed Celite® 521 was added to the product at a rate of 4 g/kg of product (31 g total) and mixed for 33 minutes. The total product volume was filtered through one filter. The final filtered volume (including buffer chase) was about 12.5 L.
  • the product pH was adjusted to 8.0 by the addition of about 566 ml of 1 M NaOH solution.
  • the final volume was about 13 L.
  • the product was filtered through one 1.2 ⁇ m Pall HDC II cartridge. This was followed by 0.65 ⁇ m Millipore® Durapore filtration (two 47 mm discs were required).
  • a Millipore® stirred cell diafiltration apparatus was assembled with a Millipore 100 kD membrane. The product was diafiltered with at least 5X volumes of EQ Buffer and concentrated to a final volume of about 112 ml.
  • the DEAE column was equilibrated with EQ Buffer.
  • Product was loaded onto the column at a flow rate of 3 ml/min. Following loading, the column was washed with about 1.4 CVs of EQ Buffer at 3 ml/min. After reversing the direction of flow of the column the column was washed with EQ Buffer +0.05 M NaCl at 2.0 ml/min for about 17.5 CVs. Elution was performed with EQ Buffer +0.2 M NaCl at a flow rate of 3.0 ml/min. Fractions (about 7.5 ml) were collected using a fraction collector. A total of 94 ml of product was pooled.
  • a total of 801 g of Cohn Fraction IV-4 paste (PN 527BE, Batch 02ZB005) was resuspended in 7201 g of water at room temperature. The resuspension was mixed for about 7 hours at 2° C. to 8° C.
  • the pH of the resuspension was adjusted to 4.9 by the addition of 80X NaAc.
  • the resuspension was centrifuged using a Sorvall® RC5B refrigerated centrifuge at 9000 rpm (15000 X g) for 8 minutes. The final supernatant volume was 7452 ml.
  • a Cuno® Biocap 1000 60LA filter capsule was flushed with 5 L of water followed by 5 L of 20 mM NaAc, pH 4.9. Acid washed Celite® 521 was added to the product at a rate of 4 g/kg of product (30 g total) and mixed for 43 minutes. The product was filtered through one Cuno® filter. A final filtrate volume (including buffer chase) was 9525 g.
  • the product pH was adjusted to 8.0 by the addition of 470 ml of 1 M NaOH solution.
  • the final volume was about 9950 ml.
  • the product was filtered through a 1.2 ⁇ m Pall HDC II cartridge. This was followed by 0.65 ⁇ m Millipore® Durapore filtration.
  • a Millipore® stirred cell diafiltration apparatus was assembled with a Millipore® 100 kD membrane. The product was diafiltered with at least 5X volumes of EQ Buffer and concentrated to a final volume of about 95 ml.
  • the DEAE column was equilibrated with EQ Buffer.
  • Product was loaded onto the column at a flow rate of 2.5 ml/min. and followed by a 4.1 CV wash with EQ Buffer at 2.5 ml/min.
  • the column was then washed with EQ Buffer +0.05 M NaCl at 1.0 ml/min for 30.5 CVs.
  • Elution was performed with EQ Buffer +0.2 M NaCl at a flow rate of 3.0 ml/min.
  • Fractions (about 7.5 ml) were collected using a fraction collector. A total of 65 ml of product was pooled.
  • MPR Master Production Record
  • Resuspension A total of 81.5 kg of Cohn Fraction IV-4 paste (PN 527BE, Batch Nos. 02ZB002 and 02ZB007) were resuspended in 738 kg of CWFI. The resuspension was mixed for 6 hours 50 minutes at 0° C. to 4° C.
  • the pH of the resuspension was adjusted to 4.90 by the addition of 1.91 kg of 80X concentrated NaAc buffer.
  • the product was adjusted to pH 8.04 by the addition of 50.34 kg of 0.4 N NaOH solution. This resulted in 1016 kg of product.
  • the product was filtered through a 30 inch 0.65 ⁇ m filter cartridge (Millipore® Durapore CVDR73TP3). Final quantity after filtration was 1005 kg.
  • a Pellicon® apparatus was assembled with 1.0 m 2 of 100 kD membranes (Millipore® PN: P2C100C05). The unit was washed with WFI and EQ buffer prior to use. The initial product pool (fractions 4 to 22 ) was concentrated to 9.82 kg and then diafiltered with 13X volumes of EQ Buffer. The final volume was 11.96 L. Later, pooled fractions 23 to 33 were concentrated and diafiltered yielding 4.3 kg. Finally fractions 34 and 35 were individually processed yielding 9.32 kg and 6.65 kg respectively. Concentrated diafiltered product resulting from fractions 23 to 35 were pooled together for further processing.
  • the column was equilibrated with a minimum of 5 CVs of EQ Buffer.
  • Concentrated diafiltered product from fractions 4 to 23 was loaded onto the column at a flow rate of 75 ml/min.
  • the column was washed with about 3 CVs (58.2 kg) of EQ buffer at 75 ml/min.
  • the column was then washed with EQ Buffer +0.05 M NaCl at 75 ml/min for about 4.7 CVs. At this point a decision was made that the concentrated diafiltered pools from fractions 23 to 35 would also be loaded onto this column.
  • the EQ Buffer+0.05 M NaCl wash was terminated and the column was re-equilibrated with 5 CVs of EQ buffer.
  • the second product pool representing fractions 23 to 35 , was loaded onto the column at a flow rate of 75 ml/min.
  • the column was washed with about 3 CVs of EQ buffer at 75 ml/min.
  • the column was then washed with EQ Buffer +0.05 M NaCl at 75 ml/min for about 41 CVs.
  • the column was eluted with a 10 CV gradient from EQ Buffer +0.05 M NaCl (low) to EQ Buffer +0.3 M NaCl (high) at a flow rate of 75 ml/min.
  • a Millipore® Pellicon apparatus with 1.0 m 2 of 100 kD membranes, was washed with WFI and EQ buffer prior to use.
  • the DEAE product pool was concentrated to 5.15 kg and then diafiltered with EQ Buffer.
  • An additional 1 liter of EQ buffer was recirculated through the Pellicon unit to recover additional product. This was repeated for a second 1 liter recirculation.
  • the final volume of concentrated diafiltered product was 8.35 kg.
  • a total of 80.0 kg of Cohn Fraction IV-4 paste (PN 527BE, Batch Nos. 02ZB004, 02ZB005, and 02ZB008) were resuspended in 720 kg of CWFI. The resuspension was mixed for 6 hours at 0° C. to 4° C.
  • the pH of the resuspension was adjusted to 4.90 (from 5.73) by the addition of 1.58 kg of 80X concentrated NaAc buffer.
  • the Cohn Fraction IV-4 resuspension was centrifuged in the Carr P18 Powerfuge at 7663 rpm at a feed flow rate of 4.0 kg/min. Temperature was maintained at 2° C. to 8° C. This resulted in 712 kg of supernatant and 17.2 kg of paste.
  • the product was adjusted to pH 7.95 by the addition of 29.78 kg of 0.4 N NaOH solution. This resulted in 883 kg of product.
  • the product was filtered through a 30 inch 0.65 ⁇ m filter cartridge. Final quantity after filtration was 839 kg.
  • the procainamide sepharose column was equilibrated with a minimum of 5 CVs of EQ buffer. Product was loaded onto the column at about 1100 ml/min with a total loading time of about 13 hours 10 minutes.
  • a Millipore® Pellicon apparatus with 1.0 m 2 of Millipore 100 kD membranes, was washed with WFI and EQ buffer prior to use.
  • the Procainamide pool was concentrated to 10.72 kg and then diafiltered with more than 16X volumes of EQ Buffer. The final diafiltered volume was 12.4 L.
  • the DEAE column was equilibrated with a minimum of 5 CVs of EQ Buffer. Diafiltered product was loaded onto the column at a flow rate of 75 ml/min. Following loading, the column was washed with about 3.3 CVs (60.4 kg) of EQ buffer at 75 ml/min. The column was then washed with EQ Buffer +0.05 M NaCl at 75 ml/min for about 77 CVs. This required about 269 hours (11.2 days). The column was eluted with a 10 CV gradient from EQ Buffer +0.05 M NaCl (low) to EQ Buffer +0.3 M NaCl (high) at a flow rate of 75 ml/min.
  • Fractions of 1000 ml were collected manually starting at the first appearance of protein as detected by a flow through UV monitor at A 280 .
  • the column was regenerated with about 4 CV of EQ +2.0 M NaCl Buffer.
  • the column was stored in 20% ethanol solution.
  • a Millipore® Pellicon apparatus assembled with 1.0 m 2 of Millipore 100 kD membranes, was washed with WFI and EQ buffer prior to use.
  • the DEAE product was concentrated to 4.75 kg and then diafiltered with 8.6X volumes of EQ Buffer.
  • An additional 1 liter of EQ buffer was recirculated through the Pellicon unit to recover additional product. This was repeated for a second 1 liter recirculation.
  • the final volume of concentrated diafiltered product was 7.8 kg.
  • Table 5 is a summary of the studies that were performed during development followed by the reasons for the modifications. TABLE 5 Fraction IV-4 Paste Resuspension Fr. IV-4 Paste Solvent Mixing Run Wt. Vol Time Temp # Lot # (g) Type (ml) pH (hr) (° C.) 1 02ZB005 800 EQ Buffer 7200 8.0 17.5 2-8 2 02ZB005 800 CWFI 7200 NA 7 2-8 3 02ZB005 801 CWFI 7201 NA 7 2-8 Resuspension, pH Adjustment, and Clarification:
  • the EQ +0.05 M NaCl wash and EQ +1.0 M NaCl elution was performed in the reverse direction relative to the load direction.
  • the elution buffer salt concentration was reduced from 1.0 M NaCl to 0.5 M NaCl in order to elute the product off the column but not other tighter binding contaminating proteins.
  • the pH of the product was adjusted from 8.05 to 4.02 by the addition of sodium acetate and acetic acid.
  • the product was 1.2 ⁇ m filtered to remove any precipitate that might result from the pH adjustment.
  • Solvent detergent mixture was added to the product and mixed overnight before loading onto the DEAE column.
  • the pooled product of the DEAE column was tested for the presence of Triton X-100. None was detected ( ⁇ about 1.0 ⁇ g/ml).
  • Total protein recovery in all the DEAE pools represented less than 0. 1% of the total protein present at the resuspension step.
  • the pH was adjusted to 4.9 by the addition of 80X Buffer (pH 4.0).
  • the volumes of 80X Buffer required were 1.91 kg for Run # 1 and 1.58 kg for Run # 2 .
  • the pH adjusted product from each lot was filtered through a single 30 inch 0.65 ⁇ m membrane filter. No signs of filter plugging were observed.
  • Procainamide Chromatography The major steps of the procainamide chromatography are summarized in Table 14. TABLE 14 Procainamide Chromatography Post Flush Wash Elution Load (EQ Buffer) (EQ + 0.075 M NaCl) (EQ + NaCl) Run CV FlowRate Vol FlowRate Vol FlowRate Vol [NaCl] FlowRate Vol # (L) (ml/min) (kg) (ml/min) (CV) (ml/min) (CV) (M) (ml/min) (kg) 1 28 1100 1005 1100 4.8 155 51.5 0.5 800 87 2 28 1100 839 1100 4.4 155 80 1.0 800 53.2
  • the BChE activity in Run # 1 continued to elute through the 2.0 M NaCi column regeneration step. It was determined that the sodium chloride concentration in the elution buffer was too low and was increased to 1.0 M for Run # 2 . As a result of the UV monitor being operated incorrectly in Run # 2 , the leading edge of the protein peak was missed and fraction collection was started late. Fortunately the “missed” BChE activity was collected with the void volume fraction. No material was lost, but the pooled volume was larger than expected. The elution fractions were assayed for BChE activity and total protein content. The results are presented graphically in FIG. 2 and FIG. 6 .
  • the original procedure for the resuspension and procainamide chromatography steps is performed at pH 8.0.
  • the pH of the product Prior to the DEAE step, the pH of the product is reduced to 4.5 to precipitate some additional protein.
  • the pH of the resuspension was reduced to 4.9 (this is the Cohn Fraction IV-5 method).
  • the pH is adjusted back to 8.0 (procainamide binding is more effective at pH 8.0).
  • the product is then filtered through 1.2 ⁇ m and 0.65 ⁇ m membrane filters before loading onto the column. This method places both precipitation steps prior to the first column.
  • the low pH treatment may also help reduce the lipid level in the product.
  • HuBChE was separated from large quantities of contaminating protein. This also suggests that it might also be possible to eliminate the lengthy 0.05 M NaCl wash by switching to a 0.0 to 0.3 M NaCl gradient. The theory being that the gradient would completely elute the low binding contaminants before eluting the HuBChE. Also, since the salt concentration of 0.3 M NaCl is higher than required to elute HuBChE, the concentration could probably be reduced to 0.2 M or 0.25 M NaCl resulting in a slight improvement in separation.
  • the present invention provides a method for the large-scale production of HuBChE.
  • “large-scale” refers to the use of starting materials, Cohn Fraction IV-4 paste in amounts greater than a few thousand grams, preferably more than about 2 kg to about 500 kg amounts, or about 2 to about 250 fold more than small-scale methods known in the art.
  • the method of the present invention uses about a 1:10 dilution of Cohn Fraction IV-4 paste to water which is then adjusted to a pH of about 4.9 with sodium acetate rather than a 1:5 dilution of paste to sodium acetate buffer.
  • the resuspension step of the present invention also differs from the prior art in that the solution is centrifuged in a continuous flow centrifuge at about 7663 rpm at a flow rate of about 4 kg/min. rather than 90 minutes at 13,700 ⁇ g. Further, depth filtration is conducted with filters rather than dialysis. Specifically, according to the present invention, the suspension is adjusted to a pH of about 8.0 and then filtered with a 0.65 ⁇ m filter cartridge. Additionally, in the method of the present invention, affinity (procainamide column) and anion exchange (DEAE) chromatography were performed only once and procainamide affinity chromatography is performed first rather than DEAE anion exchange chromatography.
  • the method of the present invention is superior to prior art methods as it results in recovery yields of about 60% having greater than about 99% purity and a specific activity of about 700 U/mg, whereas prior art methods result in yields of about 30% having about 80% purity (and up to about 90% purity using multiple washes and chromatography steps).
  • the HuBChE preparation made according to the present invention is safe, non-toxic, storage stable, highly bioavailable and proved to be an effective prophylactic treatment against OP agents.
  • the HuBChE preparation of the present invention may be administered in an effective amount to a mammal such as a human.
  • An “effective amount” is intended to mean an amount that is sufficient to treat, prevent or inhibit toxicity of an OP agent. The amount will vary depending upon factors such as the formulation and route of administration, but can nevertheless be routinely determined by one skilled in the art.
  • An “effective amount” may be readily determined by one of ordinary skill by routine methods known in the art and refers to an amount that provides an observable desired change as compared with a control.
  • compositions comprising the HuBChE preparation of the present invention may be prepared in a unit-dosage form appropriate for the desired mode of administration.
  • the pharmaceutical formulations of the present invention may be administered by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal).
  • the pharmaceutical formulations may comprise an inert, pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the pharmaceutical carrier employed may be either a solid or liquid.
  • Exemplary of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like.
  • the carrier or diluent may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible
  • Supplementary active compounds can also be incorporated into the formulations.
  • Supplementary active compounds include antibiotics, antiprotozoal agents, antifungal agents, and antiproliferative agents known in the art, analgesics and other compounds commonly used to treat exposure to OP agents and biochemical warfare agents.
  • Antibiotics include penicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin, azithromycin, clarithromycin, clindamycin, erythromycin, lincomycin, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, quinolone, cinoxacin, nalidixic acid, fluoroquinolone, ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, bacitracin, colistin, polymyxin B, sulfonamide, trimethoprim
  • Antiprotozoal agents include chloroquine, doxycycline, mefloquine, metronidazole, eplornithine, furazolidone, hydroxychloroquine, iodoquinol, pentamidine, mebendazole, piperazine, halofantrine, primaquine, pyrimethamine sulfadoxine, doxycycline, clindamycin, quinine sulfate, quinidine gluconate, quinine dihydrochloride, hydroxychloroquine sulfate, proguanil, quinine, clindamycin, atovaquone, azithromycin, suramin, melarsoprol, eflornithine, nifurtimox, amphotericin B, sodium stibogluconate, pentamidine isethionate, trimethoprim-sulfamethoxazole, pyrimethamine, sul
  • Antifungal agents include amphotericin B, fluconazole, itraconazole, ketoconazole, potassium iodide, flucytosine, and the like.
  • Antiproliferative agents such as altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene, bleomycin, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, cisplatin, cisplatin-epinephrine gel, cladribine, cytarabine liposomal, daunorubicin liposomal, daunorubicin daunomycin, dexrazoxane, docetaxel, doxorubicin, doxorubicin liposomal, epirubicin, estramustine, etoposide phosphate, etoposide VP-16, exemestane, fludarabine, fluorouracil 5-FU, fulvestrant, gemicitabine, gemtuzumab-ozogamicin, goserelin acetate, hydroxyurea, idarubicin, if
  • Supplementary compounds also include antidotes known in the art such as botulism antitoxin, tetanus antitoxin, diphtheria antitoxin, and the like.
  • the formulations of the invention may be manufactured in manners generally known for preparing pharmaceutical and cosmetic compositions, e.g., using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing.
  • the formulations may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically or cosmetically.
  • the HuBChE preparations are formulated with a carrier that will prolong the activity of the HuBChE such as a controlled release formulation, prevent or inhibit degradation or loss of activity, or prevent or inhibit loss of the HuBChE due to factors such as metabolism.
  • a carrier that will prolong the activity of the HuBChE such as a controlled release formulation, prevent or inhibit degradation or loss of activity, or prevent or inhibit loss of the HuBChE due to factors such as metabolism.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically or cosmetically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

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