U.S. patent application number 13/454029 was filed with the patent office on 2012-08-16 for pretreatment of post exposure treatment for exposure to a toxic substance by pulmonary delivery (inhaler) of a bioscavenger.
Invention is credited to Yvonne J. Rosenberg.
Application Number | 20120207738 13/454029 |
Document ID | / |
Family ID | 33551207 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120207738 |
Kind Code |
A1 |
Rosenberg; Yvonne J. |
August 16, 2012 |
Pretreatment of Post Exposure Treatment for Exposure to a Toxic
Substance by Pulmonary Delivery (Inhaler) of a Bioscavenger
Abstract
The present invention relates to a treatment by pulmonary
delivery of a bioscavenger to animals as an effective antidote to
prevent toxicity produced by exposure of an animal to nerve agents
and other toxic substances.
Inventors: |
Rosenberg; Yvonne J.;
(Washington, DC) |
Family ID: |
33551207 |
Appl. No.: |
13/454029 |
Filed: |
April 23, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11128997 |
May 13, 2005 |
8168175 |
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13454029 |
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PCT/US03/36117 |
Nov 13, 2003 |
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11128997 |
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60425726 |
Nov 13, 2002 |
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Current U.S.
Class: |
424/94.6 ;
424/94.1; 514/772; 514/777; 514/784; 514/788 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 9/0075 20130101; A61P 39/02 20180101; C12Y 301/01008 20130101;
A61K 38/465 20130101; A61K 9/0073 20130101; A61K 31/215 20130101;
A61K 38/465 20130101; A61K 45/06 20130101; C12Y 301/01007 20130101;
A61K 9/007 20130101; A61K 31/66 20130101; A61K 31/215 20130101;
A61K 2300/00 20130101; Y10S 588/901 20130101; A61K 9/0078 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/66
20130101 |
Class at
Publication: |
424/94.6 ;
424/94.1; 514/784; 514/777; 514/772; 514/788 |
International
Class: |
A61K 38/46 20060101
A61K038/46; A61K 47/12 20060101 A61K047/12; A61P 39/02 20060101
A61P039/02; A61K 47/34 20060101 A61K047/34; A61K 47/18 20060101
A61K047/18; A61K 47/22 20060101 A61K047/22; A61K 38/43 20060101
A61K038/43; A61K 47/40 20060101 A61K047/40 |
Claims
1. A method of detoxifying or neutralizing a neurotoxin or a drug
comprising administering by inhalation by an animal or a human, a
bioscavenger that prevents or eliminates the toxic effects of said
neurotoxin or said drug in said animal or said human.
2. The method of claim 1, wherein said administering is prior to
exposure of said animal or said human to said neurotoxin or said
drug.
3. The method of claim 1, wherein said administering is after said
animal or said human is exposed to said neurotoxin or said
drug.
4. The method of claim 1, wherein said neurotoxin is an
organophosphate selected from the group consisting of a nerve agent
and a pesticide.
5. The method of claim 1, wherein said bioscavenger comprises an
enzyme.
6. The method of claim 5, wherein said enzyme is homologous to said
animal or said human.
7. The method of claim 1, wherein said drug is selected from the
group consisting of cocaine, succinylcholine and heroin.
8. The method of claim 1, wherein said bioscavenger is administered
in powder form or in liquid (droplet) form.
9. The method of claim 4, wherein said organophosphate is selected
from the group consisting of sarin
(O-isopropyl-methylphosphonofluoridate), VX
(ethyl-S-2-diisopropylaminoethyl-phosphano-thiolate), MEPQ
(7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide), soman
(pinacolylmethyl-phosphonofluoridate), DFP (diisopylfluorophosphate
paraoxon), malathion and parathion.
10. The method of claim 1, wherein said bioscavenger is selected
from the group consisting of acetylcholinesterase (AChE),
carboxylesterase (CaE), paraoxonase, a bacterial organophosphate
hydrolase (OPH), a bacterial organophosphorous acid anhydride
hydrolase (OPAA) and parathion hydrolase.
11. The method of claim 1, wherein said bioscavenger is a native
molecule purified from plasma or a recombinant molecule.
12. The method of claim 11, wherein said recombinant molecule is
produced in a mammalian cell, a plant cell or an insect cell.
13. The method of claim 11, wherein said recombinant molecule is
glycosylated as in the native form of the molecule.
14. The method of claim 12, wherein said recombinant molecule is
made in a transgenic plant.
15. The method of claim 1, further comprising administering with
said bioscavenger a permeation enhancer.
16. The method of claim 15, wherein said permeation enhancer is
selected from the group consisting of oleic acid,
dimethyl-b-cyclodextrin, and citric acid and polyethylene
glycol.
17. The method of claim 3, further comprising administering an
oxime that reactivates said bioscavenger after exposure to said
neurotoxin or said drug.
18. The method of claim 17, wherein said oxime is selected from the
group consisting of 2-PAM, H16, toxogonin and TMB4.
19. The method of claim 17, further comprising administering a
permeation enhancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to U.S. provisional application
Ser. No. 60/425,726 filed Nov. 13, 2002, the contents of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a treatment by pulmonary
delivery of bioscavengers to animals as an effective antidote to
prevent toxicity produced by exposure of an animal to nerve agents
and other toxic substances. In one embodiment, the disclosure
relates to the delivery of functional bioscavenger cholinesterase
molecules as a protective in vivo treatment against poisoning by
nerve agents and drugs including but not limited to cocaine, heroin
and succinylcholine.
BACKGROUND OF THE INVENTION
[0003] Exposure to organophosphates (OPs) in the form of nerve
agents (e.g. sarin, soman and VX) and pesticides (e.g., paraoxon,
parathion and malathion) may result in acute cholinergic effects by
inhibition of acetylcholinesterase (AChE), permitting continuous
firing of neurons and thereby producing toxicity, behavioral
deficits and death. Of late, such agents pose an ever increasing
military and civilian threat due to heightened terrorist activity.
Traditional multi-drug treatment for poisoning by OPs consist of a
combination of drugs such as carbamates (e.g. pyridostigmine),
antimuscarinics, reactivators of inhibited AChE and
anti-convulsants in postexposure modalities. These treatments,
however, are far from optimal and do not prevent respiratory
stress, tremors, convulsions and behavioral impairments. In recent
years, exogenous administration of "self" native enzyme scavengers
e.g. cholinesterases (ChE) have been successfully used in many
species (mice, rats and monkeys) as safe and efficacious
prophylactic and post exposure treatments due to their capacity to
scavenge OPs in the blood and rapidly detoxify the active
components before inhibition of the endogenous targets can occur.
Such bioscavengers are shown to be highly stable, specific and
efficient, to have long half-lives in homologous systems and
capable of functioning under physiological conditions without
producing immunological or other adverse side effects. In addition
to nerve agents, certain bioscavengers can be also used to
neutralize drugs such as cocaine, heroin and succinylcholine (a
cause of apnea).
SUMMARY OF THE INVENTION
[0004] This present invention provides noninvasive and needleless
methods for the inhalation delivery of a bioscavenger capable of
rapidly ensuring entry of the bioscavenger into the blood so that
(a) a real time response to assault by a toxic substance is
accomplished, as e.g. in an incoming attack involving a nerve
agent, and (b) first responders to civilian attacks can upgrade
their protection between the time they get notification of a
chemical incident and the time they arrive on the scene. The
adaptability and portability of an inhaler also means that new
modified forms of the scavenger molecules as well as the
co-delivery of additional "enhancing" molecules can be supported in
order to increase the scavenging capabilities with reduced
treatment doses.
[0005] In one embodiment, the present invention describes a method
for the pulmonary delivery of a native or recombinant bioscavenger
for the in vivo detoxification/neutralization of organophosphates
including nerve agents, pesticides, insecticides as well as drugs
such as heroin, cocaine and succinyl choline. The bioscavenger is
administered as a single or multiple dose prophylactic
(preexposure) or therapeutic (post exposure) treatment.
[0006] In one embodiment, the present invention describes a method
for the pulmonary delivery of native or recombinant
butyrylcholinesterase for the in vivo detoxification/neutralization
of organophosphates including nerve agents, pesticides,
insecticides as well as drugs such as heroin, cocaine and succinyl
choline. The butyrylcholinesterase is administered as a single or
multiple dose preexposure or post exposure treatment.
[0007] Pursuant of the present invention, delivery of native and
recombinant (r) BChE molecules, either in powder or liquid form by
inhalation can be used for (i) Protection against chemical warfare
agents in terrorist/battlefield situations; (ii) Clinical treatment
of drug overdosing with cocaine, heroin; (iii) Alleviating life
threatening conditions such as succinylcholine-induced apnea; (iv)
neutralization or inactivation of toxic substances following pre or
post exposure of first responder civilians and farmers to nerve
agent, insecticides or pesticides. Succinylcholine is an
exogenously administered drug which causes muscle relaxation and is
given prior to surgery. This includes the muscles required for
breathing. In people lacking BChE succinylcholine cannot be
cleared, resulting in apnea (inability to breathe). This can be
overcome by treatment with BChE.
[0008] In one embodiment, the invention provides a method for the
treatment of an animal for the detoxification or neutralization of
a toxic substance which comprises administering to said animal a
bioscavenger molecule that prevents the toxic effects of said toxic
substance in the animal, wherein said bioscavenger molecule is
administered to said animal by an inhalation process. In a
preferred embodiment, the animal is a human.
[0009] In one embodiment, the bioscavenger is administered by
inhalation delivery of a dose between 1 and 10 mg per Kg of body
weight of said animal.
[0010] In another embodiment, the bioscavenger is administered by
inhalation delivery of a dose, of bioscavenger that is sufficient
to prevent the toxic effects of 2 LD50 of, e.g., a nerve agent.
[0011] In another embodiment, the invention provides single or
multiple dose preexposure administration of said bioscavenger. In
another embodiment, post exposure treatment with a bioscavenger is
given in combination with an oxime that reactivates said
bioscavenger. The bioscavenger can be a native blood-derived
product or a recombinant molecule in either a monomeric or
tetrameric form.
[0012] In a preferred embodiment, said recombinant molecules are
glycosylated in vitro to mimic the structure and function of the
native molecule.
DETAILED DESCRIPTION OF THE INVENTION
[0013] In general terms, the use of an effective pretreatment using
nerve agent scavenger such as BChE could preclude the necessity of
carrying or wearing protective clothing or masks because high
levels of the scavenger in the blood would neutralize and thus
protect against nerve agents in the absence of protective
equipment. This feature is important to soldiers in the Army in
battlefield conditibns and to the Marines who guard Embassies
worldwide and who are unable to don protective clothing. In terms
of non-military personnel, treatment with bioscavengers is
important to any civilian first responders who must enter an
exposed area, those exposed to environmental toxins in insecticides
and those suffering drug overdose and apnea.
[0014] Based on availability, broad spectrum efficacy and safety,
the cholinesterase, butyrylcholinesterase (BChE) is the only pan
scavenger candidate sufficiently developed for human treatment and
is the preferred bioscavenger of the present invention. A pan
scavenger is one which works on multiple nerve agent targets. Since
cholinesterases are stoichiometric inhibitors (one molecule of
enzyme neutralizing one molecule of nerve agent), humans require a
large dose of scavenger e.g. 150-200 mg (.about.3 mg/Kg) of BChE,
in order to protect against an exposure of 2 LD.sub.50 of nerve
agent. While bioscavengers can be administered via IM, IV,
transdermally or by pulmonary routes prior to exposure, an inhaler
is by far the simplest, safest and most efficient means of
delivery. Traditionally, bioscavenger drugs/treatments have been
administered orally or via the intramuscular route using
autoinjectors. Major limitations to the use of the commonly used
modes of delivery is the inability to deliver large molecules
(transdermal patches), a long delay in reaching blood peak levels
of bioscavenger activity, major soreness at the injection sites and
potential infections (intramuscular) and the impracticality in
battlefield/high risk conditions of intravenous injections. By
contrast, pulmonary administration of peptides and proteins can be
expected to lead to higher and more rapid rates of systemic
absorption than other non-invasive routes because the alveolar
epithelium where absorption takes place is thin and has a large
surface area (.about.1,500 sq fl). Pulmonary delivery is performed
via introduction of the bioscavenger through the nose or mouth via
inhaler or nebulizer. In a preferred embodiment, delivery is by
mouth with an inhaler.
[0015] A critical feature required of any effective nerve agent
scavenger is that as a pre or post exposure treatment, it must (i)
have good stability, that is, circulate in the blood at high
concentrations for prolonged periods and (ii) in emergency
conditions, the enzyme must reach peak levels as quickly as
possible. To prevent toxicity, nerve agents must be reduced to a
level below their median lethal dose within one blood circulation
time.
[0016] Scavenger enzymes are usually complex glycoproteins and
stability of the molecules is greatly influenced by glycosylation
profiles, efficiency of folding and multimerization. In this
regard, unlike the native blood-derived forms, recombinant
prophylactic/therapeutic molecules produced by genetic engineering
e.g. BChE, exhibit microheterogeneity in the sugar residues which
negatively impacts on the rate of clearance in vivo and may limit
their use as human treatments. In the present invention, this
deficiency is overcome by in vitro glycosylation methodologies
which complete or correct sugar profiles of the "incorrectly"
expressed recombinant protein and produce a form of the scavenger
which mimics the native form in structure and pharmacokinetic
function. As therapeutic human treatments, "remodeled sialyted"
recombinant molecules, in contrast to native plasma-derived
molecules, should not suffer from batch to batch variability or
from potential safety issues associated with contaminating
infectious agents (HIV-1, hepatitis, prions, etc.)
[0017] It is understood that the present invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein, as these may be substituted or altered without
deviating from the invention, and will be understood by one of
ordinary skill in the art. It is also to be understood that the
terminology used herein is used for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention. It must be noted that as used herein and
in the appended claims, the singular forms "a," "an," and "the"
include plural reference unless the context clearly dictates
otherwise.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Prevention of toxic effects of a toxic substance "in" an animal is
meant to include any toxic effects that may manifest "on" the
animal, as in the case for toxic effects on the skin or other
exposed surface. Preferred methods, devices, and materials are
described, although any methods and materials similar or equivalent
to those described herein can be used in the practice or testing of
the present invention. All references cited herein are incorporated
by reference herein in their entirety.
[0019] In a preferred embodiment, the invention provides methods
for the delivery of a homologous bioscavenger. In the treatment of
humans, an example of a homologous bioscavenger would be the human
BChE product, which can be blood derived or recombinant. In the
absence of an available homologous bioscavenger, a single treatment
with heterologous can be performed. If repeated administrations are
required as in long term pre-exposure protection, then it must be
homologous because the body would make an immune response to a
foreign protein and eliminate the protein from the body.
[0020] The bioscavenger can be administered in a powder or liquid
droplet form.
[0021] It is one object of the present invention to deliver a
variety of scavenger molecules including but not limited to native
self enzyme molecules including, but not limited to,
(butyrylcholinesterase (BChE), acetylcholinesterase (AChE)),
carboxylesterase (CaE), paraoxonase, and bacterial enzymes such as
organophosphate hydrolases (OPH), organophosphorous acid anhydride
hydrolases (OPAA) and parathion hydrolase. For optimal efficiency,
native molecules must be homologous with the recipients (Example
3). Life Sciences. Vol 72, p125 2002.
[0022] It is another object of the present invention to deliver
scavenger molecules that have been successfully used as a safe and
efficacious treatments to prevent poisoning by organophosphate (OP)
compounds in the form of nerve agents including but not limited to
organophosphates such as sarin
(O-isopropyl-methylphosphonofluoridate),VX
(ethyl-S-2-diisopropylaminoethy1-phosphano-thiolate), MEPQ
(7-(Methylethoxyphosphinyloxy)-1-methylquinon-linium iodide), soman
(pinacolylmethl-phosphonofiumidate), DFP=diisopylfluorophosphate
paraoxon, malathion and parathion.
[0023] In one preferred embodiment, the bioscavenger is
administered as a tetramer. In another embodiment, the bioscavenger
is administered as a monomer. In some embodiments, the bioscavenger
is a recombinant molecule. The bioscavenger molecule can be a
recombinant molecule produced in vitro in mammalian or insect
cells, or in transgenic plants or livestock.
[0024] In one embodiment, the invention provides a method of
inhalation delivery of an altered bioscavenger which comprises a
mutation with increased bioscavenging efficacy, for example as in
E197Q BChE or E202Q AChE mutations. In another embodiment, the
invention contemplates the post-exposure co-administration of the
bioscavenger with an oxime which reactivates said bioscavenger. In
some preferred embodiments, the oxime is selected from the group
consisting of 2-PAM, 1116, toxogonin, TMB4.
[0025] The present invention provides an efficient, highly
manageable and user-friendly means of delivery by inhalation
(puffer) of bioscavenger molecules e.g. BChE, AChE in sufficient
amounts required for protection against toxicity by nerve agents,
insecticides and drugs. In the case of a stoichiometric (i.e.,
"BChE-like") bioscavenger alone, .about.150-200 mg (.about.3 mg/Kg)
is required to protect against an exposure of 2 LD.sub.50 of nerve
agent.
[0026] The methods disclosed herein encompass the use of inhalers
to administer either powder or liquid forms of the bioscavenger,
depending on the chemical properties of the candidate molecules
(see Example 1). In one preferred embodiment, the bioscavenger is
administered prophylactically, i.e., over a period of several weeks
prior to any anticipated or possible exposure to the toxic agent.
It is presently contemplated that a preferred dosage is delivered
at a rate of .about.10 puffs from a powder inhaler (.about.15
mg/puff of BChE) and that this dosage will initially protect
against high levels of toxin exposure within 30 minutes. In a
preferred embodiment, the treatment is repeated about 10 times on
the first day, with maintenance puffs thereafter sufficient to
provide protection.
[0027] In another embodiment, the invention provides methods of an
initial administration of a bioscavenger rapidly across the
pulmonary epithelium, as in the case of an impending (i.e, within
30-60 minutes) exposure to the toxic substance. In this embodiment,
a truncated monomer bioscaveriger molecule is administered so that
the bioscavenger is more rapidly enters the blood.
[0028] It is one object of the present invention to develop an
adaptable, noninvasive and needleless delivery system capable of
rapidly ensuring entry of the bioscavenger into the blood so that
(a) a real time response to an incoming attack would not be
unreasonable, (b) civilian first responders could upgrade their
protection between the time they get notification of a chemical
incident and the time they arrive on the scene and (c) an injured
soldier/victim can easily receive passive bioscavenger delivered by
an another soldier/associate.
[0029] As a receptor or soluble receptor, the bioscavenger agent
binds, sequesters, and clears the toxin as a complex from the body.
As an enzyme, the agent binds, inactivates by hydrolytic or
non-hydrolytic processes, resulting in toxins that are no longer
harmful to mammalian tissues and/or are removed more rapidly from
the host. Inactivation can occur, but is not limited to enzymatic
cleavage, blocking of reactive moieties, masking of active site(s),
sequestering to certain tissues, and/or clearance of the toxin as a
bound or unbound complex.
[0030] It is another object of the present invention to deliver
scavenger molecules that have been successfully used as a safe and
efficacious anti-toxicants of nerve agents as both pre-exposure and
post-exposure treatments.
[0031] It is another object of the present invention to deliver
scavenger molecules that have been successfully used as a safe and
efficacious post-exposure treatment to overcome drug overdosing
such as cocaine and heroin.
[0032] It is another object of the present invention to deliver
scavenger molecules that have been successfully used as a safe and
efficacious post-exposure treatment to prevent apnea induced by the
muscle relaxant succinylcholine.
[0033] Transpulmonary administration of peptides and proteins can
be expected to lead to higher rates of systemic, absorption because
of a large surface area (.about.1,500 sq ft). However,
transpulmonary administration of high molecular weight compounds is
almost always incomplete, because of absorption barriers in the
alveolar epithelium. Permeation enhancers can increase the
bioavailibility of inhaled peptides and proteins in the blood by
for example, increasing the paracellular permeability through tight
junctions, a mechanism that depends on Ca++ channels.
[0034] In one embodiment of the present invention, protection
against agent or drug toxicity is achieved using pulmonary delivery
of any form of the scavengers in combination with permeation
enhancers including but not limited to oleic acid,
dimethyl-b-cyclodextrin and citric acid and polyethylene glycol
(PEGylation).
[0035] The methods disclosed herein encompass the delivery of
homologous recombinant bioscavenger molecules. The scavenger genes
in question are cloned into the appropriate mammalian or insect
cell expression vector (Example 4) or plant expression vector
(Example 5). It is contemplated in the present invention that a
transgenic construct of interest can be delivered to mammalian or
plant cells by viral-mediated or non-viral mediated means.
Recombinant virus vectors utilized in the present invention
include, but are not limited to (I) retroviral vectors, including
but not limited to vectors derived from a Moloney murine leukemia
virus (MoMLV) or a myeloproliferative sarcoma virus (MPSV) (ii)
adenovirus vectors (iii) adeno-associated vectors (iv) herpes
simplex virus vectors (v) SV40 vectors (vi) polyoma virus vectors
(vii) papilloma virus vectors; (viii) picornavirus vectors; and,
(ix) vaccinia virus vectors. Depending on the virus vector system
chosen, techniques available to the skilled artisan are utilized to
infect the target cell of choice with the recombinant virus
vector.
[0036] In some embodiments, these scavenger molecules are highly
active complex tetrameric, glycoproteins e.g., BChE produced by the
co-expression Of a peptide containing a proline-rich attachment
domain (PRAD) in the expressing cells with the cholinesterase gene
(Example 7). Alternatively, PRAD protein can be added in vitro to
monomeric and dimeric forms of the scavenger molecules to effect
tetramerization.
[0037] The methods disclosed herein encompasses methods for
production of recombinant bioscavenger molecules in transgenic
plants (Example 5). a mammalian cell system in vitro e.g. CHO
(Example 6) as well as in the breast milk of transgenic livestock
e.g. pigs, cattle and sheep.
[0038] In one preferred embodiment,. the methods disclosed herein
encompass a device for the delivery of homologous native (blood
derived) BChE molecules that are purified, where appropriate, by
procainamide and DEAE chromatography and administered alone or in
combination with other molecules. In one embodiment, these
scavenger molecule are complex tetrameric, glycoproteins such as
butyrlcholinesterase (EC3.1.1.8 acylcholine achydrolase,
pseudocholin-esterase, non-specific cholinesterase), a serine
esterase (MW=345,000) comprised of four identical subunits
containing 574 amino acids and held together by non-covalent bonds
and contains 36 carbohydrate chains (23.9% by weight).
[0039] In addition to tetrameric molecules, monomeric BChE may be
generated by inserting a stop codon at G534. Thus the mutant
monomeric molecule produced lacks the 41 C-terminal residues and
thus the functional tetramerization domain required for tetramer
formation and in vivo stability (Example 8). The monomer has
several advantages despite the fact that its stability in plasma is
very poor. In addition to the >8-fold increase in activity per
ml of CHO supernatant, a monomer may be able to more rapidly cross
the blood-brain barrier and exhibit much higher bioavailablity in
the plasma following delivery via inhalers than the larger
tetrameric molecules. In one embodiment, monomeric molecules,
despite being ineffective in maintaining long term protection
compared to the highly stable tetramers, may be highly efficacious
in emergency situations that require real time responses and rapid
treatment or booster administrations.
[0040] As a complex recombinant glycoprotein, a scavenger may
require additional post-translational modifications to enable the
agent to provide the necessary disabling function(s) similar to the
native protein. Such glycoproteins are often produced with either
incomplete or wrong sugar profiles compared to their plasma derived
counterparts. For example: 1) The lack of a functional
a,2,6-sialyltransferase (ST) gene in CHO cells 2). The presence of
xylosidayed- and fucosylated-type sugar chains in many
plant-derived glycoproteins and the absence of sialic acid in
plants 3). The presence of galactosyl transferases in pigs
resulting in the potential surface expression of a, 1.3 galactose,
which is not normally found in humans. Several approaches that are
available to overcome these innate deficiencies have either
involved exposing recombinant proteins in vitro to enzymes such as
exoglycosidases and sialyltransferases (Example 9) or introducing
liver-derived enzyme beta-galactoside alpha-2, 6-sialyltransferase
cDNA by gene transfer into those cells producing the recombinant
protein. The in vitro incorporation of sialic acid into recombinant
proteins (developed specifically to allow efficient sialic acid
capping of beta-galactose-exposed termini) has been highly
successful. Such findings are in agreement with data showing that
liver (the in vivo source of many of these highly sialylated
glycoproteins) contain sialyltransferase, involved in the
sialylation of O-glycosidically linked carbohydrate chains on serum
glycoproteins. The in vitro glycosylation methodology utilized to
modify the recombinant bioscavenger molecule can include, but is
not limited to, glycosylation where the recombinant protein
preparation is incubated with appropriate enzymes in solution or
coupled to a solid support. These enzymes include but are not
limited to, glycosltransferases, such as sialtransferases,
galactotransferases, and fucosyltransferases.
[0041] In one preferred embodiment, following pulmonary delivery,
improved pharmacokinetic profiles (stability) and manufacturing
efficiencies of tetrameric and monomeric scavenger molecules,
either wild type or mutant, produced in the various expression
systems is achieved following in vitro sialylation of the
recombinant glycoproteins to "correct" the microheterogeneity in
their glycosylation profiles.
[0042] The rate of detoxification of an OP by a bioscasvenger
enzyme molecule is determined by three parameters: (I) the rate of
inhibition of the enzyme by the OP (ii) the rate of aging of the
OP-inhibited enzyme and (iii) the rate of reactivation of the
enzyme by oximes. Following interaction of the OP with the
scavenger enzyme, the latter may become immediately inhibited or
undergo spontaneous or oxime-induced reactivation. In the latter
case, the reaction of oxime nucleophile with the phosphonylated
enzyme leads to displacement of the phosphonyl group and
restoration of normal activity. In one embodiment of the present
invention bioscavenger inhalation treatments can be co-administered
post exposure with specific oxime molecules which can reactivate
the enzyme scavenger and thus reducing the amount of scavenger
required (Example 10). Such oxime molecules include but are not
limited to 2-PAM, HI6, toxogonin, TMB4. This is particularly
important, since many potent enzyme scavengers are stoichiometric
inhibitors and require large amount of protein for protection
(150-200 mg BChE per adult). In one embodiment the present
invention provides mutant BChE clones (e.g. E197Q BChE or E202Q
AChE) with a slower rate of aging and thus potentially superior
scavenging capability that are delivered by inhalation as another
means of reducing the amount of enzyme required for protection. In
one embodiment of the present invention, a combination of mutant
BChE plus oxime is delivered post-exposure via the lungs to further
enhance the scavenging efficacy of nerve agent antidotes.
[0043] In general, the process of inhalation is meant to encompass
the concept of delivery of a substance to the blood via the lungs,
wherein delivery takes place across the pulmonary epithelium of an
animal. Inhalation can be via mouth, nose or intratracheal. Most
inhalers use the mouth, which is a preferred method of inhalation
in the present invention. Delivery by inhalation can be by means of
an inhaler or nebulizer, many of Which are known to those of
ordinary skill in the art. See, e.g., U.S. Pat. No 6,595,202 the
contents of which are incorporated by reference herein. Delivery by
an inhaler is preferred. Delivery can be an active process of the
animal to which the bioscavenger is administered or via a passive
means. Some pulmonary delivery techniques rely on the inhalation of
a pharmaceutical formulation by the patient so that the active drug
within the dispersion can reach the distal (alveolar) regions of
the lung. A variety of aerosolization systems have been proposed to
disperse pharmaceutical formulations. For example, U.S. Pat. Nos.
5,785,049 and 5,740,794, the disclosures of which are herein
incorporated by reference, describe exemplary powder dispersion
devices which utilize a compressed gas to aerosolize a powder.
Other types of aerosolization systems include those which typically
have a drug that is stored in a propellant, nebulizers which
aerosolize liquids using a compressed gas, and the like.
[0044] Many of the advantages of administering BChE by inhalation
include the following: non-invasive; user friendly; suitable for
repeated administration; can deliver small and large
molecules/proteins and peptides; large absorptive surface area for
delivery; highly permeable single cell membrane; and rapid
(depending on compound).
[0045] The following examples further illustrate experiments that
have demonstrated reduction to practice and utility of selected
preferred embodiments of the present invention, although they are
in no way a limitation of the teachings or disclosure of the
present invention as set forth herein.
[0046] Other objectives, features and advantages of the present
invention will become apparent from the following specific
examples. The specific examples, while indicating specific
embodiments of the invention, are provided by way of illustration
only. Accordingly, the present invention also includes those
various changes and modifications Within the spirit and scope of
the invention that may become apparent to those skilled in the art
from this detailed description.
EXAMPLES
Example 1
[0047] Below are the relevant properties of complex molecules which
currently determine whether liquid or powder inhalers are
chosen:
Liquid
[0048] 1. Inhalers are expensive
[0049] 2. Most proteins are made in a liquid form, thus lower
development costs for some molecules
[0050] 3. Must be kept frozen or cold. RT prior to use
[0051] 4. 50 ul/puff (at 20mg/ml=1 mg puff)
[0052] 5. .about.65% get to the lung=.about.650 ug
[0053] 6. Require >200 puffs for 150-200 mg
[0054] 7. Bioavailability (lung to blood, depending on the
compound)
Powder
[0055] 1. Inhalers are not as expensive
[0056] 2. Must produce a powder. May be difficult and less
efficient to make a large molecule into 1-4 .mu. particles
[0057] 3. High stability as a powder at RT
[0058] 4. Up to 25-30 mg/puff
[0059] 5. .about.50% gets to the lungs=.about.15 mg
[0060] 6. Require 10-15 puffs for 150-200 mg
[0061] 7. Bioavailability (lung to blood, depending on the
compound)
Example 2
[0062] The importance of Using Homologous Scavenger Molecules in
Vivo
[0063] As bioscavengers, molecules must exhibit good
bioavailability and good stability (high mean retention times, MRT)
in blood following administration. Work in monkeys using purified
native macaque BChE (MaBChE), has shown that homologous BChE has a
very long retention time in blood (MRT =225+/-19 hours following a
single i.v. injection) compared to current treatments and induces
no antibody responses. By comparison, an injection of heterologous
human (Hu)BChE into monkeys results in a short retention time (MRT
33.7 +/-2/9 hours) and induces antibodies. In addition, the
administration of 7,000 U (10 mg) of purified homologous macaque
BChE into macaques is known to protect against 2.1 LD50'of VX and
3..about.LD50 soman with no induction of anti-BChE antibody and no
adverse toxicological effects. The development of a human treatment
require evidence in monkeys of similar stability following
pulmonary delivery. Life Sciences. Vol 72, p12 2002.
Example 3
The Effect of Absorption Enhancers on the Bioavaiiability of
Bioscavengers in the Blood Following Pulmonary Delivery
[0064] Oleic acid, dimethyl-b-cyclodextrin and citric acid are
initially tested at different concentrations. 1,500-2,000 units of
MaBChE are mixed with 50 or 250 ug of the different enhancers and
the enzyme activity (bioavailability) in the blood is assessed at
various times following intracheal administration into the mid
lung.
Example 4
Cloning of MaBChE Gene in Mammalian Cell Expression Vector
[0065] For the production of rMaBChE in mammalian or insect cells,
plants, transgenic animals and/or insects, the BChE gene from liver
of macaques obtained from National Primate Research Center has been
cloned. Total RNA was isolated from the liver and cDNA was
synthesized by reverse transcriptase with oligo dT as primer. The
synthesized DNA oligonuclotide primers used for the amplification
of MaBChE gene from the cDNA were based on the human BChE
sequence.
[0066] First the MaBChE gene was PCR amplified in two fragments, 5'
and 3' fragments using PfuTurbo DNA Polymerase (Stratagene), and
the amplified fragments were cloned into pCRH vector (Invitrogen).
The 5' fragment was amplified with a pair of primers, O-Pro#11 and
Pro#5, and the 3' fragment with O-Pro#4 and Pro#12. The resulting
vectors containing the nucleotide sequences encoding
NH.sub.2-terminal and COOH-terminal fragments of MaBChE were
digested with appropriate restriction enzymes and cloned into
pcDNA3.1 (Invitrogen) to form a single MaBChE reading frame
(pcDNA3.1-MaBChE). The genes cloned in pcDNA3.1 are expressed under
the control of CMV promoter and the cells transfected with the
vector are to be selected with G418. The nucleotide sequence of
macaque BChE gene in the constructed vectors was confirmed by
commercial sequencing.
Example 5
Cloning of MaBChE Gene in Plant Expression Vector and Enzyme
Production
[0067] The production of MaBChE in plant or plant cells requires a
plant-specific expression vector. For the optimal activity of the
recombinant BChE, after production in plant, the protein is
chemically glycosylated in the pattern mimics the pattern of
endogenous glycosylation. When the proteins are expressed in plant
via secretion pathway, they are heavily glycosylated in a plant
specific manner which is problematic to remove for the chemical
gycosylation. One means to prevent plant specific glycosylation is
to design a vector which expresses the transgene in the endoplasmic
reticulum and then complete glycosylation in vitro.
[0068] For the plant expression vector construct, one useful vector
is the pTRAkt plant vector. The nucleotide gene sequence encoding
mature MaBChE is PCR amplified utilizing PfuTurbo DNA Polymerase
from a previously constructed plasmid vector, pcDNA3.1-MaBChE, with
a pair of primers containing appropriate restriction enzyme
sequences. The resulting about 1.8 kb amplified fragment is cloned
into an intermediate pCRII vector, The nucleotide sequence of the
cloned gene in pCRII vector is sequenced by commercial DNA
sequencing. The MaBChE gene is excised from the pCRII vector with
appropriate restriction enzymes and cloned into pTRAkt.
Agrobacterium-Mediated Transient Expression System and Plant
Transformation
[0069] Agrobacteria is transformed with each of the plant
expression vectors by electroporation. Recombinant Agrobacteria
harboring MaBChE gene is coinfiltrated with the Agrobacteria
harboring PRAD fragment into tobacco leaves by vacuum application.
After infiltration, leaves are incubated adaxial side down, on
wetted paper in sealed trays at 23.degree. C. with a 16 h
photoperiod. After 60 h, leaves are frozen in liquid nitrogen and
stored at 80.degree. C. until analyzed. Transient expression by
agro-infiltration of tobacco leaves is highly efficient with
accumulation levels being similar to those found in transgenic
plants. Stable proteins yields up to 20-40 mg/kg fresh plant
material have been obtained. For plant transformation, either
tobacco leaf disks or YT-2 suspension cells are co-cultivated with
recombinant agrobacteria, placed on selective media and regenerated
to intact plant or further cultivated as suspension cells.
Protoplast Preparation
[0070] Protoplasts of suspension cells are isolated enzymatically
using Cellulase and Pectyolase. The cells are incubated in the
enzyme solution, filtered from cellular debris and then washed with
buffer. Protoplasts are resuspended in a medium that favors
elongative growth and cultured in the dark.
Extraction of Proteins from Infiltrated Leaves
[0071] For the extraction of transiently expressed recombinant
proteins infiltrated leaves are ground in liquid nitrogen to a fine
powder with a mortar and pestle. Soluble protein is extracted with
extraction buffer, cell debris is removed by two rounds of
centrifugation, and the supernatant is used for expression analyses
and further protein purification by affinity chromatography or
sucrose gradient.
Purification of Protein Extracts from Infiltrated Leaves by
Affinity Chromatography
[0072] Soluble protein is extracted with extraction buffer, cell
debris is removed by two rounds of centrifugation. A Ni-NTA column
is equilibrated with binding buffer, and leaf extract is applied to
the column at a constant flow rate. After sample application, the
column is washed with binding buffer. Nonspecifically bound
proteins are removed with binding buffer containing 25 mM
imidazole. His6-tagged gp120 is eluted by using binding buffer
containing 250 mM imidazole.
Agrobacterium-Mediated Transient Expression Systems
[0073] Agrobacteria is transformed with each of the plant
expression vectors by electroporation. Recombinant Agrobacteria is
infiltrated into tobacco leaves by vacuum application. After
infiltration, leaves is incubated adaxial side down, on wetted
paper in sealed trays at 23.degree. C. with a 16h photoperiod.
After 60 h, leaves are frozen in liquid nitrogen and stored at
80.degree. C. until analyzed. For transient expression in tobacco
suspension cultures, cells are co-cultivated with recombinant
Agrobacteria on agar plates first, then transferred to liquid media
and incubated for another two days. After harvesting the cells can
be frozen and stored until further processed and analyzed.
Purification of Protein Extracts from Infiltrated Leaves by
Affinity Chromatography
[0074] Soluble protein is extracted with extraction buffer, cell
debris is removed by two rounds of centrifugation. A Ni-NTA column
is equilibrated with binding buffer, and leaf extract is applied to
the column at a constant flow rate. After sample application, the
column is washed with binding buffer. Nonspecifically bound
proteins is removed with binding buffer containing 25 mM imidazole.
Tetrameric MaBChE is eluted by using binding buffer containing 250
mM imidazole.
Example 6
MaBChE Production in CHO-K1 Cells
[0075] Establishment of CHO cells that continuously produces and
expresses primate (monkey or human) BChE demonstrates the principle
of this invention. CHO cells were used that were stably transduced
with a CMV or retroviral vector in which the BChE gene is driven by
the long-terminal repeat regulatory region. For the production of
MaBChE, CHO-K1 cells were transfected with pcDNA3.1-MaBChE vector
using LIPOFECTAMINE PLUS reagent (Invitrogen) by the manufacture's
procedure. Two days after transfection, G418 sulfate was added to
the cell at the concentration of 400mg/liter for the transfected
cells. After 2 weeks selection, single cell colonies were prepared
by limiting dilution for the isolation of cells expressing high
level of MaBChE. When cells reached near confluence, the cell media
was changed to fresh media and the cells were allowed to secrete
MaBChE for 2 days. The 2-day media was collected and the BChE
activity measured. The BChE expressed was tested to be biologically
active. Out of 20 single cell-derived transfected colonies, 3
colonies showed higher than 0.2 unit/ml.
[0076] These expressing cells were then adapted to grow in
suspension in CHO-S-SFM (serum-free media). High cell densities,
typically 2.0.times.10.sup.6 cells/ml were obtained from spinner
flask cultures. Partial purification of BChE from CHO cell cultured
media revealed that the level of impurities in SFM was
significantly lower than the serum-supplemented DMEM. This suggests
that additional steps need not be employed in the purification of
butyrlcholinesterase from SFM. This would result in a reduction of
the operating time by 50 h and boost the recovery yield of BChE to
75%.
[0077] To confirm the existence of transgene in the cells
expressing high level of MaBChE, genomic DNA was isolated from
cells of each colony. MaBChE nucleotide sequence in the isolated
genomic DNA was detected by the PCR amplification using MaBChE
specific oligonucleotides.
Example 7
[0078] Coexpression of a PRAD Peptide Together with
Butyrlcholinesterase in Mammalian and Plant Cells Expressing Bche
Enhances Tetrameric Forms of the Enzyme
[0079] The principle of this invention is further demonstrated by
the ability to enhance tetramerization of expressed monomeric
butyrlcholinesterase expressed in mammalian and plant cells by the
co-expression of a proline-rich attachment domain (PRAD).
[0080] Data suggest that for optimal detoxification activity by
BChE, the tetrameric form of the enzyme is required. A heteromeric
form of BChE is, formed by the attachment of the catalytic subunit
to a triple helical collagen-like tail subunit. The function of the
collagen-like tail is to anchor catalytic subunits to the basal
lamina. The triple helical association of three collagen-like
strands, ColQ, forms the tail. The PRAD of each strand can bind the
catalytic subunit tetramer producing the asymmetric moieties.
[0081] The PRAD fragment was amplified using PfuTurbo DNA
Polymerase and a pair of synthesized oligonucleotide primers,
O-Pro#6 and O-Pro#8, and the amplified fragments were cloned into
pCRII vector (Invitrogen). A plasmid vector of PRAD with FLAG
sequence (PRAD-FLAG) at the carboxyterminal in pCRII vector was
also amplified using PfuTurbo DNA Polymerase and a pair of
synthesized oligonucleotide primers, O-Pro#6 and O-Pro#7 and the
amplified fragment was cloned into pCRII vector. The addition of
FLAG at the carboxyterminal PRAD will allow the purification of
MaBChE tetramer by passing the culture fluid over a FLAG-antibody
containing column. The nucleotide sequence of both PRAD and
PRAD-FLAG has been confirmed by commercial sequencing of the
fragments in the vectors. The nucleotide sequence encoding PRAD and
PRAD-FLAG fragments are excised from the corresponding vectors with
appropriate restriction enzymes and cloned into pcDNA4 plasmid
vector for mammalian cell expression, and pTRAkt plasmid vector for
plant expression.
[0082] For the production of tetrameric BChE, CHO-K1 tranfected
with pcDNA3.1-MaBChE and expressing high level of MaBChE is
retransfected with pcDNA4-PRAD or pcDNA4-PRAD-FLAG. The transfected
cells are selected by the addition of zeocine in the media at a
concentration of 200 mg/liter for 2 weeks. The amount of tetrameric
MChE relative to dimer and monomer are determined by northern blot
of the media collected after 2 days secretion of BChE using
anti-BChE antibody. If necessary, high ratio of tetramer producing
cells are isolated by limiting dilution of the transfected
cells.
Example 8
[0083] Cloning of MaBChE-534stop Encoding Bche Monomeric
Molecules
[0084] A plasmid vector harboring truncated form of MaBChE which
prevents tetramerization was made for the production of momomeric
MaBChE production in mammalian cells. The DNA fragment lacking the
sequence encoding the tertramerization domain was prepared by PCR
amplification of MaBChE gene using PfuTurbo DNA Polymerase with a
pair of oligonucleotide primers, O-Pro#11 and O-Pro#33. The
resulted DNA fragment, MaBChE-534stop was cloned into an
intermediate pCRII vector. The nucleotide sequence of the truncated
MaBChE was confirmed by commercial sequencing. The MaBChE-534stop
DNA fragment was excised from the pCRII vector with appropriate
restriction enzymes and cloned into pcDNA (pcDNA3.1-MaBChE-534stop)
for the expression in mammalian cells. For the expression in plant,
the MaBChE-534stop fragment is cloned into pTRAkt.
Example 9
[0085] In Vitro Post-Translational Modification of
Butyrlcholinesterase to Produce a Recombinant Protein with
Properties Similar to the Native Form
[0086] As previously noted, the native glycosylation profile of any
effective nerve agent scavenger is essential for good in vivo
stability and while many expression systems have been very
successful in expressing functional non-gylcosylated proteins, they
have been inadequate in terms of preserving the correct glycans on
heavily glycosylated proteins such as BChE. Even though clever
molecular engineering and other elegant manipulations of producer
cells, animal or plants are largely overcoming these problems, they
have often met with limited success to date because they are
imperfect, time consuming and may sacrifice expression levels. An
alternate and much more rapid technology is the sialylation of the
expressed purified protein in vitro. The structures with exposed
GlcNAc and/or Galactose with remodeling resulting in nearly
quantitative coverage of all galactosyl residues by sialic acid.
This technology has to date been use to successfully remodel >40
compounds and can results in an increase in the number of
sialylation sites occupied from of 64% to 92%.
Example 10
The Ability of the OP-Inhibited RbChEs to Undergo Spontaneous or
Oxime-Induced Reactivation
[0087] Due to rapid irreversible inhibition of OP-inhibited ChEs,
reactivation of the enzyme scavenger is often virtually impossible.
This is particularly the case following interaction with nerve
agents such as soman which renders the enzyme non-reactivatable
almost immediately. At a mechanistic level, the reaction between
organophosphates and ChEs results in the creation of phosphylated
enzyme complexes involving the active-center serine (S198 for BChE)
followed by either spontaneous or induced regeneration of the
active site. Reaction of OP with the BChE-esterase results in rapid
cleavage of the alkoxy-O-P bond and the formation of
P--O.sup.--conjugates resulting in irreversible inhibition "aging"
of some enzyme scavengers. Studies on aging of AChE have been shown
to depend on the structure of the OP, enzyme source, pH,
temperature and ionic strength of the solution. The aging process,
characteristic of ChEs (in contrast to carboxylesterase)
bioscavengers, has stimulated the generation of ChE mutants which
are more easily reactivated than the wild type enzyme. For example,
E202Q, an AChE mutant has been shown to be 2-3 times better in
detoxifying sarin and soman, decreased the affinity of soman for
AChE, slowed the reactivation of soman-inhibited AChE by HI-6 and
decreased aging. In the presence of 2 mM of HI-6, the same amount
of WT and E202Q AChE could detoxify 135- and 225.about.fold molar
excess of soman respectively, indicating the superior properties of
the mutant compared to the WT enzyme.
[0088] In the latter case, the reaction of oxime nucleophile with
the phosphonylated enzyme leads to displacement of the phosphonyl
group and restoration of normal activity. Thus, oximes act on the
inactivated ChE rather than protect against OP inactivation itself.
In this context, exogenously administered FBS AChE inhibited by the
powerful anticholinesterase MEPQ has been shown to be reactivated
in mice by an i.m. injection of TMB4 (1,1.trimethylene bis
(4-hydroxyhiminiomethylpyridinium) permitting the reactivated
enzyme to protect the mice against exposure to an additional dose
of MEPQ.
Example 11
[0089] Cloning of E197Q BChE Mutant Gene with Enhanced Scavenging
Ability
[0090] In an attempt to increase scavenging efficacy, mutants
enzyme molecules can be created which exhibit reduced rates of
inhibition following interaction with OP as well as a reduced rate
of aging, thus allowing persistence of the active enzyme. In this
context, an amino acid change, replacement of 197 glutamic acid to
glutamine (E197Q), of hrBChE has shown better toxic agent
scavenging activity. A plasmid vector harbouring the E197Q mutation
in MaBChE (pcDNA3.1-MaBChE-E197Q) has been constructed by
site-directed mutagenesis. First, E197Q point mutation containing
oligonucleotides (O-Pro#49 and O-Pro#10) were synthesized from both
strands of MaBChE gene in pcDNA3.1-MaBChE plasmid. Two fragments of
MaBChE gene were PCR amplified using PfuTurbo DNA Polymerase and
two pairs of primers. The 5'-fragment was amplified by O-Pro#11 and
O-Pro#10 and the 3'-fragment by O-Pro#9 and O-Pro#12. The resulting
DNA fragments were isolated from the reaction mix by PCR
purification kit (Invitrogen). The amplified and purified 5'- and
3'-fragments were mixed and 5 thermal-cycles were performed in the
reaction mix without primers to create the full length template.
The resulted full length template containing E197Q mutation, then,
diluted 100 to 1000 fold and was used as a template to amplify the
E197Q MaBChE gene using PfuTurbo DNA Polymerase with O-Pro#11 and
O-Pro#12. The amplified fragment was cloned into an intermediate
pCRII vector and the nucleotide sequence was confirmed by
commercial sequencing. The MaBChE-E197Q gene in pCRII vector was
excised by appropriate restriction enzymes and cloned into pcDNA3.1
for the expression in mammalian cells.
* * * * *