U.S. patent application number 11/007695 was filed with the patent office on 2005-07-21 for method for treating, preventing, or inhibiting enterotoxigenic escherichia coli infections with bovine red blood cells.
Invention is credited to Cassels, Frederick J., Ryu, Hyoik.
Application Number | 20050158284 11/007695 |
Document ID | / |
Family ID | 27732723 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158284 |
Kind Code |
A1 |
Ryu, Hyoik ; et al. |
July 21, 2005 |
Method for treating, preventing, or inhibiting enterotoxigenic
escherichia coli infections with bovine red blood cells
Abstract
Disclosed herein are red blood cell (RBC) preparations and
methods of making and using thereof for treating, preventing or
inhibiting enterotoxigenic Escherichia coli (ETEC) infections. In
particular, bovine RBC preparations are shown to reduce ETEC
adhesion when administered orally. Also disclosed are kits
comprising RBC preparations for the treatment of ETEC
infections.
Inventors: |
Ryu, Hyoik; (Daegu, KR)
; Cassels, Frederick J.; (Ellicott City, MD) |
Correspondence
Address: |
OFFICE OF THE STAFF JUDGE ADVOCATE (SKS)
U.S. ARMY MED. RESEARCH & MATERIAL COMMAND
504 SCOTT STREET
ATTN: MCMR-JA (MS. ELIZABETH ARWINE)
FORT DETRICK
MD
21702-5012
US
|
Family ID: |
27732723 |
Appl. No.: |
11/007695 |
Filed: |
December 9, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11007695 |
Dec 9, 2004 |
|
|
|
10077804 |
Feb 20, 2002 |
|
|
|
6869602 |
|
|
|
|
Current U.S.
Class: |
424/93.7 ;
514/159; 514/253.08; 514/304 |
Current CPC
Class: |
A61K 35/18 20130101;
A61K 2300/00 20130101; A61K 35/18 20130101 |
Class at
Publication: |
424/093.7 ;
514/304; 514/253.08; 514/159 |
International
Class: |
A61K 045/00; A61K
031/496; A61K 031/46; A61K 031/60 |
Claims
1. An oral formulation for treating, preventing, or inhibiting an
enterotoxigenic Escherichia coli (ETEC) infection comprising a
therapeutically effective amount of at least one red blood cell
(RBC) preparation and a carrier suitable for oral
administration.
2. The formulation of claim 1, further comprising at least one
supplementary active compound.
3. The formulation of claim 2, wherein the supplementary active
compound is an anti-diarrheal or an antibiotic.
4. The formulation of claim 3, wherein the supplementary active
compound is bismuth subsalicylate, diphenoxylate with atropine,
loperamide HCl, attapulgite, or ciprofloxacin.
5. The formulation of claim 4, further comprising a composition
that binds LT.
6. The formulation of claim 1, wherein the carrier is an ingestible
carrier.
7. The formulation of claim 1, wherein the carrier is a
pharmaceutical carrier or a foodstuff.
8. The formulation of claim 1, wherein the ETEC infection is
travelers' diarrhea or infant diarrhea.
9. The formulation of claim 1, wherein the RBC preparation is a
bovine RBC preparation.
10. The formulation of claim 1, wherein the RBC preparation
comprises whole erythrocytes, erythrocyte ghosts, erythrocyte
fractions, erythrocyte extracts, glycolipid receptors isolated from
erythrocytes, or combinations thereof.
11-24. (canceled)
25. A kit for the treatment, prevention, or inhibition of an
enterotoxigenic Escherichia coli (ETEC) infection comprising the
formulation of claim 1 packaged together with directions for
use.
26. The kit of claim 25, wherein the formulation is provided as
individual doses.
27. The kit of claim 25, further comprising a food, a foodstuff, or
a food supplement.
28. The kit of claim 25, further comprising a supplementary active
compound.
Description
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0001] This invention was made by employees of the United States
Army. The government has rights in the invention.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to bovine erythrocyte
preparations. In particular, the present invention relates to
methods of using bovine erythrocyte preparations for treating,
preventing, or inhibiting enterotoxigenic Escherichia coli
infections.
[0004] 2. Description of the Related Art
[0005] Diarrhea caused by enterotoxigenic Escherichia coli (ETEC),
commonly referred to as travelers' diarrhea, is a common health
problem among travelers visiting less developed or tropical
countries. See Peltola, H., et al. (1991) Lancet 338: 1285-1289 and
Ericsson, C. D. et al. (1993) Clin. Infect. Dis. 16: 616-626.
Diarrhea caused by ETEC and other ETEC infections are important
concerns for military personnel when deployed to less developed
countries. See Wolf, M. K., et al. (1993) Clin. Microbiol. 31:
851-856 and Bourgeois, A. L., et al. (1993) Am. J. Trop. Med. Hyg.
48: 243-248. ETEC may be transmitted by food or water contaminated
with animal or human feces. ETEC produces two toxins, a heat-stable
toxin (ST) and a heat-labile toxin (LT). ETEC infections may cause
profuse watery diarrhea, abdominal cramping, fever, nausea,
vomiting, chills, loss of appetite, headache, muscle aches, and
bloating.
[0006] The current therapy for travelers' diarrhea is to initiate
treatment with agents such as bismuth subsalicylate
(Pepto-Bismol.RTM.), antidiarrheals such as diphenoxylate with
atropine (Lomotil.RTM.), loperamide HCl (Immodium.RTM.),
attapulgite (Kaopectate.RTM.) and the like, rehydration therapy,
and combinations thereof. The majority of the treatments involve
the non-specific removal of the offending agents (i.e. toxins) from
the intestinal tract. Only in moderate to severe cases of diarrhea
where distressing or incapacitating symptoms are reported is
antimicrobial therapy recommended. ETEC is frequently resistant to
common antibiotics such as trimethoprim-sulfamethoxazole and
ampicillin. Fluoroquinolones such as ciprofloxacin have shown some
efficacy. Antibiotics are not usually effective at reducing
clinical symptoms of the disease and problems associated with
antibiotic resistance can occur. Prophylactic use of antibiotics is
not recommended. Thus, therapies that specifically remove ETEC from
the intestine are needed to provide more effective treatments for
ETEC diarrhea.
[0007] In order to initiate the infectious process of diarrhea,
ETEC must adhere to the host intestinal epithelial cells via the
binding between bacterial adhesins and host receptors. This binding
is commonly referred to as adhesion-receptor interaction. See
Beachey, E. H. (1981) J. Infect. Dis. 143: 325-345; Satterwhite, T.
K., et al. (1978) Lancet. 2: 181-184; and Warner, L. and Y. S. Kim.
(1989) "Intestinal Receptors for Microbial Attachment", Eds. M. J.
G. Farthing, and G. T. Kensch, ENTERIC INFECTION: MECHANISMS,
MANIFESTATIONS AND MANAGEMENT, pp. 31-40. Raven Press, New York.
One treatment method that is based on this adhesin-receptor
interaction, described by U.S. Pat. No. 5,897,860 ('860 patent),
involves an oligosaccharide covalently attached to a solid support,
wherein the oligosaccharide binds E. coli heat-labile toxin. The
'860 patent does not prevent or treat ETEC infections as the
methods merely immobilize or neutralize the heat-labile toxin (LT).
Thus, the enterotoxigenic E. coli are still capable of adhering to
the intestinal epithelial cells, colonizing and producing more
LT.
[0008] Thus, a need still exists for a method for treating,
preventing, or inhibiting ETEC infections, diseases, or disorders
such as travelers' diarrhea.
SUMMARY OF THE INVENTION
[0009] The present invention generally relates to erythrocyte (RBC)
preparations, such as bovine erythrocyte (bRBC) preparations and
methods using thereof for treating, preventing, or inhibiting
enterotoxigenic E. coli (ETEC) infections.
[0010] In some embodiments, the present invention relates to a
formulation for treating, preventing, or inhibiting an
enterotoxigenic E. coli (ETEC) infection, such as travelers'
diarrhea or infant diarrhea, comprising a therapeutically effective
amount of at least one RBC preparation and a carrier suitable for
oral administration. The formulation may further comprise at least
one supplementary active compound such as an anti-diarrheal or an
antibiotic. Preferred supplementary active compounds include
bismuth subsalicylate, diphenoxylate with atropine, loperamide HCl,
attapulgite, and ciprofloxacin. The formulation may further include
a composition that binds LT. In some preferred embodiments, the
formulation comprises a carrier such as an ingestible carrier. The
carrier may be a pharmaceutical carrier or a foodstuff.
[0011] In some preferred embodiments, the RBC preparation is a
bovine RBC preparation. The RBC preparation may comprise whole
erythrocytes, erythrocyte ghosts, erythrocyte fractions,
erythrocyte extracts, glycolipid receptors isolated from
erythrocytes, or combinations thereof.
[0012] In some embodiments, the present invention relates to a
method of treating, preventing, or inhibiting an ETEC infection in
a subject comprising administering to the subject a formulation
comprising a therapeutically effective amount of at least one RBC
preparation and a carrier suitable for oral administration. The
method may further comprise administering to the subject at least
one supplementary active compound such as a supplementary active
compound is an anti-diarrheal or an antibiotic. Preferred
supplementary active compounds include bismuth subsalicylate,
diphenoxylate with atropine, loperamide HCl, attapulgite, and
ciprofloxacin. In some embodiments, the method may further comprise
administering to the subject a composition that binds LT.
[0013] In some embodiments, the present invention relates to a
method of preventing, inhibiting, or reducing an amount of
enterotoxigenic E. coli (ETEC) adhering to a subject's intestinal
wall comprising administering to the subject a formulation
comprising a therapeutically effective amount of at least one RBC
preparation and a carrier suitable for oral administration. In some
embodiments, the amount of ETEC adhering to the subject's
intestinal wall is prevented, inhibited, or reduced by about 10% or
greater, preferably about 20% or greater, more preferably, about
30% or greater, more preferably, about 40% or greater, most
preferably, about 50% or greater.
[0014] In some embodiments, the present invention relates to a kit
for the treatment, prevention, or inhibition of an enterotoxigenic
Escherichia coli (ETEC) infection comprising a formulation
comprising a therapeutically effective amount of at least one RBC
preparation and a carrier suitable for oral administration packaged
together with directions for use. The kit may comprise the
formulation in individual doses. In some embodiments, the kit may
comprise a food, a foodstuff, or a food supplement. The kit may
further comprise a supplementary active compound.
[0015] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are intended to provide further
explanation of the invention as claimed. The accompanying drawings
are included to provide a further understanding of the invention
and are incorporated in and constitute part of this specification,
illustrate several embodiments of the invention and together with
the description serve to explain the principles of the
invention.
DESCRIPTION OF THE DRAWINGS
[0016] This invention is further understood by reference to the
drawings wherein:
[0017] FIG. 1. Adherence of a human ETEC M424C 1 (CS1, CS3) to
rabbit small intestinal microvilli (n=3).
[0018] FIG. 2. Removal of a human enterotoxigenic Escherichia coli
strain M424C 1 (CS1, CS3) attached to rabbit small intestinal
microvilli by bovine erythrocytes (n=2). Agglutination scores: BRBC
No. 1, 4+; BRBC No. 2. 1+.
[0019] FIG. 3. Lipid profiles of bRBC and rabbit intestinal tissue
extracts by high performance thin layer chromatography.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention generally relates to erythrocyte (RBC)
preparations and methods using thereof for treating, preventing, or
inhibiting enterotoxigenic E. coli (ETEC) infections. As used
herein "RBC preparations" include whole erythrocytes, erythrocyte
ghosts, erythrocyte fractions, erythrocyte extracts, glycolipid
receptors isolated from RBCs, and combinations thereof. As used
herein "erythrocyte fractions" means any part of the erythrocyte
rather than the intact whole erythrocyte and includes soluble
extracts, debris, ghosts, and the like. As used herein "ETEC
infections" include infections caused by ETEC such as travelers'
diarrhea, infant diarrhea, and the like. In preferred embodiments,
the RBC preparations are bovine RBC (bRBC) preparations which
include whole bovine erythrocytes, bovine erythrocyte ghosts,
bovine erythrocyte fractions, bovine erythrocyte extracts,
glycolipid receptors isolated from bovine RBCs, and combinations
thereof.
[0021] As disclosed herein, adhesion of ETEC to host epithelial
cells may be prevented, inhibited, or reduced by binding to RBC
preparations such as bRBC preparations. Specifically, prevention,
inhibition, or reduction of ETEC adhesion may be achieved by
competitive binding to bRBC preparations as the binding affinity
between ETEC and bovine erythrocytes is greater than the binding
affinity between ETEC and intestinal epithelial cells.
Consequently, ETEC infections may be treated, prevented, or
inhibited by outcompeting the adhesion of ETEC to intestinal
epithelial cells with the greater binding affinity of ETEC to bRBC
preparations, by adsorbing off adherent ETEC from the host
epithelial cell receptors with bRBC preparations, or a combination
thereof. Thus, the present invention provides a method of treating,
preventing, or inhibiting an ETEC infection in a subject comprising
administering to the subject at least one bRBC preparation.
[0022] Examples of ETEC strains that are known to adhere to and
agglutinate bovine erythrocytes (comprising receptors) include
strains expressing colonization factors (CF, the adhesion protein)
such as CFA/I, CFA/III, CS1, CS2, CS4, CS5, CS7, CS15, CS17, CS19,
PCF0159 and PCF0166. See Cassels, F. J., and M. W. Wolf (1995) J.
Ind. Microbiol. 15: 214-226; De Graaf, F. K., and F. R. Mooi (1986)
Adv. Microb. Physiol. 28: 65-143; Evans, D., Jr., et al. (1979)
Infect. Immun. 23: 336-346; Khalil, S. B., et al. (1999) Infect.
Immun. 67: 4019-4026; and Ryu, H., et al. (2001) Infect. Immun. 69:
640-649, which are herein incorporated by reference.
[0023] The ability of the RBC preparations of the present invention
to prevent, inhibit, or reduce adhesion of ETEC to the intestinal
wall of a subject may be measured by any of the methods available
to those skilled in the art; including in vitro and in vivo assays.
See e.g. Evans, D., et al. (1979) Infect. Immun. 23: 336-346;
Foldhar, J. (1995) Methods Enzymology 253: 43-50; Gonzolez, E. A.,
et al. (1985) FEMS Microbiol. Lett. 29: 115-121; and Knutton, S.,
et al. (1984) Infect. Immun. 44: 519-527, which are herein
incorporated by reference. Examples of suitable assays for activity
measurements are provided herein. Properties of the preparations
may be assessed, for example, by using one or more of the assays
set out in the Examples below or those known in the art. Other
pharmacological methods conventional in the art may be used to
determine the efficacy of the RBC preparations for treating,
preventing, or inhibiting ETEC infections.
[0024] The RBC preparations in accordance with the present
invention are useful in the treatment of ETEC infections,
preferably travelers' diarrhea, infant diarrhea, and the like. The
RBC preparations of the present invention may be used in
combination with or as a substitution for treatments of ETEC
infections and related conditions. For example, the RBC
preparations may also be used alone or in combination with at least
one supplementary active compound such as bismuth subsalicylate,
diphenoxylate with atropine, loperamide HCl, attapulgite,
ciprofloxacin, and the like to treat, prevent or inhibit ETEC
infections. Additionally, the RBC preparations may be used alone or
in combination with compositions which bind E. coli heat-labile
toxin (LT) such as those described in U.S. Pat. No. 5,891,860,
which is herein incorporated by reference.
[0025] A RBC preparation of the present invention may be
administered in a therapeutically effective amount to a mammal such
as a human. Therapeutically effective amounts of the RBC
preparations may be used to treat, prevent, or inhibit ETEC
infections. An "effective amount" is intended to mean that amount
of a RBC preparation that, when administered to a subject, is
sufficient to prevent, inhibit, or reduce adhesion of ETEC to the
intestinal wall of the subject or reduce the severity of symptoms,
e.g. from severe to moderate. A "therapeutically effective amount"
of a given RBC preparation, is a quantity sufficient to treat,
prevent, or inhibit ETEC infections in a subject. The amount of a
given RBC preparation that will correspond to such an amount will
vary depending upon factors such as the particular RBC preparation,
the formulation and route of administration, the ETEC strain, and
the identity of the subject being treated, but can nevertheless be
routinely determined by one skilled in the art. Also, as used
herein, a "therapeutically effective amount" of a given RBC
preparation of the present invention is an amount that prevents,
inhibits, or reduces the amount of ETEC adhering to the intestinal
wall of a subject as compared to a control by routine methods known
in the art.
[0026] For example, a therapeutically effective amount of a RBC
preparation of the invention ranges from about 0.001 to about 1,000
mg total dry weight per kg body weight, preferably about 0.001 to
about 500 mg total dry weight per kg body weight, and more
preferably about 0.001 to about 250 mg total dry weight per kg body
weight. The skilled artisan will appreciate that certain factors
may influence the amount of the RBC preparation required to
effectively treat a subject, including but not limited to the
severity of the ETEC infection, previous treatments, the general
health and/or age of the subject, and other diseases present.
[0027] Moreover, treatment of a subject with a therapeutically
effective amount of the RBC preparation preferably includes a
single treatment, but can include a series of treatments. For
example, a subject may be treated with a RBC preparation of the
invention at least once. However, the subject may treated with the
RBC preparation from about one time per week to about once daily
for a given treatment period. The length of the treatment period
will depend on a variety of factors such as the severity of the
ETEC infection, the period of exposure to ETEC, the risk of ETEC
infection, and the like. It will also be appreciated that the
effective dosage of the RBC preparation used for treatment may
increase or decrease over the course of a particular treatment.
Changes in dosage may result and become apparent by standard
diagnostic assays known in the art. In some instances chronic
administration may be required. The RBC preparation may be
administered before ETEC exposure or infection, during ETEC
exposure or infection, after ETEC exposure or infection, or a
combination thereof.
[0028] The RBC preparations of the invention can be incorporated
into formulations suitable for the intended route of
administration. Preferred routes of administration include oral
administration. Formulations suitable for oral administration are
well known in the art. The formulations of the present invention
comprise a therapeutically effective amount of at least one RBC
preparation and an inert, carrier or diluent. As used herein, the
carrier or diluent may be an ingestible carrier or a
pharmaceutically acceptable carrier. Suitable ingestible carriers
include food, foodstuffs, and food supplements, such as red meat,
milk, protein drinks, and nutritional sports bars. Pharmaceutically
acceptable carriers include solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are compatible with oral
administration. The 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. Similarly, 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. Except insofar as any
conventional media or agent is incompatible with RBC formulations,
use thereof in the formulations is contemplated.
[0029] Supplementary active compounds can also be incorporated into
the formulations. Supplementary active compounds include bismuth
subsalicylate, diphenoxylate with atropine, loperamide HCl,
attapulgite, ciprofloxacin, and the like commonly used to treat
ETEC infections. Additionally, compositions that bind LT such as
those described in U.S. Pat. No. 5,891,860, may be incorporated
into the formulations.
[0030] In preferred embodiments, the RBC preparation of the present
invention is formulated to be compatible with oral administration.
A variety of formulations can be employed. Thus, if a solid carrier
is used, the formulation can be tableted, placed in a hard gelatin
capsule in powder or pellet form or in the form of a troche or
lozenge. The amount of solid carrier may vary, but generally will
be from about 25 mg to about 1 g. If a liquid carrier is used, the
preparation will be in the form of syrup, emulsion, soft gelatin
capsule, or suspension in an ampoule or vial.
[0031] The formulations of the invention may be manufactured in
manners generally known for preparing pharmaceutical formulations
and food supplements, e.g., using conventional techniques such as
mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing. For oral
administration, the RBC preparations can be formulated readily by
combining the RBC preparations with carriers known in the art such
as those used in pharmaceuticals or nutritional supplements. Such
carriers enable the RBC preparations of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
subject to be treated. Formulations for oral use can be obtained
using a solid excipient in admixture with at least one RBC
preparation, optionally grinding the resulting mixture, and
processing the mixture of granules after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients include: fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; and cellulose
preparations, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may
be added, such as crosslinked polyvinyl pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate.
[0032] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally comprise gum arabic, polyvinyl pyrrolidone, Carbopol
gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compounds and agents.
[0033] Oral formulations of the present invention include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of
gelatin and a plasticizer, such as glycerol or sorbitol. The
push-fit capsules can comprise the RBC preparation of the present
invention in admixture with fillers such as lactose, binders such
as starches, and/or lubricants such as talc or magnesium stearate,
and, optionally, stabilizers. In soft capsules, the RBC preparation
may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration. Thus, the
RBC preparations of the invention are preferably prepared in a
unit-dosage form appropriate for oral administration.
[0034] Oral formulations generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the RBC preparation can be incorporated with
excipients and used in the form of a tablet, a bolus, a troche, or
a capsule which may comprise any of the following ingredients, or
compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring. Some preferred
formulations for oral formulations include microcrystalline
tablets, gelatin capsules, or the like.
[0035] Additionally, the compounds may be delivered using a
sustained-release system, such as semi-permeable matrices of solid
hydrophobic polymers comprising the RBC preparation. Various
sustained-release materials have been established and are known by
those skilled in the art. Sustained-release capsules may, depending
on their chemical nature, release the RBC preparation for a few
weeks up to over 100 days. Depending on the chemical nature and the
biological stability of the RBC preparation, additional strategies
for protein stabilization may be employed. The formulations also
may comprise suitable solid- or gel-phase carriers or excipients.
Examples of such carriers or excipients include calcium carbonate,
calcium phosphate, sugars, starches, cellulose derivatives,
gelatin, and polymers such as polyethylene glycols.
[0036] In some embodiments, the RBC preparations are prepared with
carriers that will protect the RBC preparations against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. 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 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.
[0037] It is especially advantageous to formulate oral compositions
in dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the subject to be
treated; each unit comprising a predetermined quantity of a RBC
preparation calculated to produce the desired therapeutic effect in
association with the required carrier. The specification for the
dosage unit forms of the invention are dictated by and directly
dependent on the unique characteristics of the RBC preparation and
the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such a RBC
preparation for the treatment of individuals. Preferred
formulations include bRBC preparations in an enteric coated capsule
that releases the bRBC preparation into the small intestine of a
subject where the interactions with ETEC would occur.
[0038] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0039] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
RBC Ghost Preparation
[0040] RBC preparations were made according to a modified procedure
described by Dodge et al. (1962) Arch. Biochem. Biophys. 201:
119-130, which is herein incorporated by reference. Specifically,
15 ml of blood in acid-citrate-dextrose solution (ACD) (Sigma
Chemical, Co., St. Louis, Mo.) was placed in a 50 ml plastic (or
glass) tube and centrifuge at 1,300 g (Sorvall RC-3 with HL-8
rotor, at 2000 rpm) for 15 minutes. The Buffy coat (a white layer
primarily comprising lymphocytes formed between plasma (top) and
packed RBC (bottom) when blood in an anticoagulant such as ACD) was
first centrifuged, and then the supernatant fluid containing serum
was aspirated off.
[0041] Then the RBC pellet was washed up to four times by filling
the tube with normal saline (0.9% NaCl in H.sub.2O, 9 g/L),
resuspending the RBC by gently rocking the tube for several times
and centrifugation as before. The RBC pellet was washed until the
supernatant was clear, but no more than 4 times. Then the RBC
pellet was resuspended in isotonic PBS, 50 mM, pH. 7.4 (1 volume of
sodium phosphate monobasic solution (NaH.sub.2PO.sub.4, 0.155 M,
18.6 g/L) to 6 volumes of sodium phosphate dibasic solution
(Na.sub.2HPO.sub.4.7H.sub.2O, 0.103 M, 27.6 g/L)), and incubated in
an ice bath for 30 minutes. After the ice bath incubation, the RBC
solution was centrifuged as before and the supernatant was
aspirated off.
[0042] Three (3) ml of the packed RBC was transferred to a 40 ml
Nalgene tube (Nalge Nunc International, Rochester, N.Y.) containing
30 ml of a hypotonic buffer solution (1:16 dilution of isotonic PBS
in H.sub.2O), pH. 7.4, and then mixed on a Thermolyne Varimix
(Barnstead/Thermolyne, Dubuque, IA) for 15 minutes at room
temperature followed by centrifugation at 25,000 g (Sorvall RC-5B
with SS-34 rotor; at 14,400 rpm) for 15 minutes. The supernatant
was aspirated off and the pellet (RBC ghost) was resuspended,
washed in the hypotonic buffer solution, and centrifuged three
times. Then the supernatant was aspirated off and the pellet was
stored in -70.degree. C. until further use.
[0043] It is noted that 4 of 10 liters of blood obtained from a cow
yielded 540 ml of packed RBC ghosts (about 3.8 g total protein).
Thus, large scale production could comprise obtaining about 500 ml
of blood per week per each cow in a herd of about 200 cattle (or
about 2-5 sacrifices) per year would provide economically feasible
amounts of RBC preparations.
EXAMPLE 2
Agglutination Assays
[0044] Tables 1-4 below show the agglutinating properties of bRBC
preparations with human ETEC strains. For each, ETEC strains were
grown on colonization factor antigen (CFA) agar (Becton Dickinson,
Sparks, MD) or CFA agar comprising 0.15% bile salts (Becton
Dickinson, Sparks, Md.) at 37.degree. C. overnight and the bacteria
were harvested in phosphate buffered saline (PBS, pH 7.4). The
bacterial concentration was adjusted to 10.times.of 0.5 optical
density at 600 nm for standardization, 2% bRBC was prepared in
phosphate buffered saline (PBS) comprising 50 mM .alpha.-methyl
mannopyranoside (Sigma Chemical, Co., St. Lois, Mo.). For the slide
hemagglutination assays, 8 .mu.l of the ETEC suspension was mixed
with the same volume of 2% RBC on a microscopic slide for 2
minutes. The degree of hemagglutination was observed visually and
graded based on the clump size and time of agglutination (see score
of agglutination, Table 3).
[0045] Table 1 shows the ability of bRBC to agglutinate human ETEC
strains.
1TABLE 1 Hemagglutinating activity of various ETEC strains with
bovine erythrocytes ETEC Strain Colonization Factor
Hemagglutination H10407 CFA/I.sup.+ + H10407P CFA/I.sup.- -
H10407NM CFA/I.sup.+ + M452C1 CFA/I.sup.+ + Z26-5 CFA/III.sup.+ +
60R75 CS 1.sup.+ + M424C1 CS 1.sup.+, CS 3.sup.+ + C91f CS 2.sup.+
+ E9034A CS 3.sup.+ .+-. E8775 CS 4.sup.+, CS 6.sup.+
Autoagglutination E11881B CS 4.sup.-, CS 6.sup.- - E11881E CS
4.sup.+, CS 6.sup.+ - E17018A CS 5.sup.+, CS 6.sup.+ + E17018B CS
5.sup.-, CS 6.sup.- - PE423 CS 5 - D02-2 CS 7.sup.+ + E20738A CS
17.sup.+ + E20738B CS 17.sup.- - C95-1059 CS19 + Z78-6 PCF
0159.sup.+ + G723 PCF 0159.sup.- - Z163-3 PCF 0166.sup.+ +
[0046] Table 2 shows the ability of bRBC and its ghost preparation
to agglutinate several ETEC strains.
2TABLE 2 Agglutination of a bRBC and fractions by several ETEC
strains ETEC Strain E11881E M424C1 60R75 H10407NM H10407P Fraction
CS4, CS6 CS1, CS3 CS1 CFA/I CFA/I negative Hemagglutination with
RBC - + + + - RBC ghosts - + + + -
[0047] Table 3 shows differences in hemagglutination with human
ETEC strains between RBCs from two individual cows.
3TABLE 3 Differences in hemagglutination with human ETEC strains
between erythrocytes from two individual cows ETEC strain Cow
number* (Colonization Factor) No. 8884 No. 9654 H10407 NM (CFA/I)
3** 0 60R75 (CS1) 4 3 C91f (CS2) 3 0 M424C1 (CS1, CS3) 4 3 E17018A
(CS5, CS6) 3 2 E20738A (CS17) 4 4 Z163-3 (PCF0166) 3 0 *USDA Bovine
Mastitis Laboratory Designation (Beltsville, MD). **Score of
agglutination: 4, agglutination < 10 sec; 3, agglutination 10-30
sec; 2, agglutination 0.5-1 min; 1, agglutination 1-2 min; 0, no
agglutination within 2 min.
[0048] Table 4 compares the agglutinating ability of intact RBC and
its ghost preparation with human ETEC strains.
4TABLE 4 Agglutination of intact bovine erythrocyte (Cow No. 8884)
and its ghost preparation with human ETEC strains ETEC strain
(Colonization Factor) Whole bRBC bRBC ghost H10407 NM (CFA/I) 3* 3
60R75 (CS1) 4 3 C91f (CS2) 3 3 M424C1 (CS1, CS3) 4 3 E17018A (CS5,
CS6) 3 3 E20738A (CS17) 4 4 Z163-3 (PCF0166) 3 2 *Score of
agglutination: 4, agglutination < 10 sec; 3, agglutination 10-30
sec; 2, agglutination 0.5-1 min; 1, agglutination 1-2 min; 0, no
agglutination within 2 min.
EXAMPLE 3
RBC Preparation Stability
[0049] The stability of the RBC ghost preparations was tested by
the slide agglutination assay described in Example 2. The RBC ghost
preparations were stored at -20.degree. C. and -70.degree. C. About
every two to four months, the preparations were thawed and used for
the agglutination assay. The remaining preparations were frozen and
stored again at -20.degree. C. and -70.degree. C.
[0050] The repeated freezing and thawing up to 5 times did not
alter the original agglutination activity of the ghost preparation.
Thus, storage of the RBC ghost preparation at -20.degree. C. and
-70.degree. C. did not alter the original agglutination activity as
compared to the intact bRBC preparations.
EXAMPLE 4
Analysis of Glycolipid Receptors Isolated from bRBC Ghosts
[0051] Glycolipids and phospholipids were extracted from the bRBC
ghosts by methanol:chloroform:water (10:5:3) extraction. The lipid
profile of the RBC ghost was analyzed by high performance thin
layer chromatography (HPTLC) and adherence of human ETEC strains to
the lipid extraction of RBC ghost was examined by HPTLC overlay
procedure with .sup.14C-labeled ETEC strain. See Magnani, J. et al.
(1982) JBC 257 :14365, which is herein incorporated by reference.
As show in the HPTLC orcinol plate, the RBC ghost was found to
contain several glycolipids and phospholipids, such as asialo-GM2,
asialo-GM1, GM1, lactosylceramide and phosphyatidylethanolamine,
that are well-known receptors for many human ETEC strains. See FIG.
3, see also Karlsson, K. A. (1989) Ann. Rev. Biochem. 58: 309-350,
which is herein incorporated by reference. Also several ETEC
strains (E9034A, 60R315, C91 f, and the like) were found to bind
the lipid extract of bRBC by the HPTLC overlay procedure.
EXAMPLE 5
Absorptive Capacity and Competition Assay
[0052] To examine the absorptive capacity of bRBC, 2% intact bRBC
(diluted to 0.1% and 0.01% total volume) was mixed with an
optically adjusted ETEC H10407 (2.2.times.10.sup.9 bacteria/ml)
suspension and incubated at 37.degree. C. for two minutes (Table
5). At 2, 5, 10, 20, and 60 minutes after incubation, 0.1 ml was
taken out of the bactera-bRBC mixture followed by centrifugation at
1,300 g for 2 minutes at room temperature to remove the adherent
bacteria to bRBC. The supernatant fluid (free suspended bacteria)
was serially diluted 1:10 and inoculated on CFA agar plates in
duplicates followed by overnight incubation at 37.degree. C. See
e.g. Cheney, C. P., et al. (1979) Infect. Immun. 26: 736-743, see
also Wenneras, C., et al. MOLECULAR PATHOGENESIS OF
GASTROINTESTINAL INFECTIONS. Plenum Press, NY (1991), pp. 327-330,
both of which are herein incorporated by reference. Table 5 shows
the ability of erythrocytes to remove ETEC strains in vitro.
Specifically, it was found that mixing intact RBC, 0.1% and 0.01%
total volume, with a CFA/I bearing H10407 suspension that the
bacterial concentration was reduced by 59% and 57% after 2 minutes
incubation, respectively since the numbers of the free suspended
bacteria at 0.1% and 0.01% bRBC after removal of the adherent
bacteria to bRBC were 41% and 43% the control (unabsorbed with
bRBC), respectively.
5TABLE 5 Reduction of E. coli H10407 (CFA/I) colony counts by bRBC
Number of bacteria* Incubation Time (minutes) 0.1% RBC 0.01% RBC 2
9.1 .times. 10.sup.7 (41%) 9.5 .times. 10.sup.7 (43%) 5 1.1 .times.
10.sup.8 (49%) 1.7 .times. 10.sup.8 (76%) 10 1.1 .times. 10.sup.8
(50%) 1.7 .times. 10.sup.8 (76%) 20 9.8 .times. 10.sup.7 (45%) 1.3
.times. 10.sup.8 (60%) 60 3.5 .times. 10.sup.8 (159%) 5.6 .times.
10.sup.8 (255%) *Control: 2.2 .times. 10.sup.8 bacteria/ml.
[0053] For the competition assay, rabbit brush borders (RBB) were
prepared according to Ryu, H. et al. (2001) Infect. Immun. 69:
640-649, which is herein incorporated by reference. A bacterial
adherence assay was conducted by mixing 50 .mu.l of RBB (1 mg/ml
protein), 30 .mu.l of PBS and 20 .mu.l of an ETEC strain, M424C1
(CS1,3), which binds to RBB in a glass test tube in duplicates.
After incubation for 15 minutes at room temperature on a rotating
platform, the tubes were mixed vigorously for 10 seconds on setting
5 with a Vortex-Geni (Scientific Industries, Bohemia, MY). The
bacteria-RBB mixture was washed twice with PBS by centrifugation,
at 1,100 g for 10 minutes and the pellet was gently resuspended in
100 .mu.l of PBS. Ten (10) .mu.l of 2% bRBC and PBS were added to
the competition assay tube and control tube, respectively, followed
by incubation for 5 minutes at room temperature on a rotating
platform. The bacterial adherence to RBB was examined at 1,000
power in oil immersion utilizing Nomarski differential interfacee
phase contrast optics (Olympus USAH2). FIGS. 1 and 2 show the
ability of bovine erythrocyte ghosts to remove the adherent
bacteria from the RBB microvilli. When examined under a phase
contrast microscope, the RBC ghosts attached to ETEC that were
bound to the brush borders, and appeared to be competing off
RBB-bound bacteria away from the RBB.
EXAMPLE 6
In vivo Assay in Human Subjects
[0054] To determine whether a given RBC preparation is effective in
preventing, inhibiting, or treating a disease or infection
associated with enterotoxigenic E. coli, the following assay may be
conducted. To determine whether the given RBC preparation is
effective to present or treat an ETEC disease or infection, the RBC
preparation is administered to subjects before or during the
challenge with the ETEC strain. To determine whether the given RBC
preparation is effective to treat an ETEC disease or infection, the
RBC preparation is administered after the onset of symptoms of ETEC
infection after challenge with the ETEC strain.
[0055] An ETEC strain, such as H10407, may be used. See Levine, M.
M., et al. (1982) Scand. J. Infect. Dis. 33: 83-95; Levine, M. M.,
et al. (1979) Infect. Immun. 23: 729-736;
[0056] Levine, M. M., et al. (1980) Am. J. Epidemiol. 111: 347-355;
and Tacket, C. O., et al. (1988) N. Engl. J. Med. 318: 1240-1243,
which are herein incorporated by reference. To a liquid culture of
the ETEC strain, glycerol is added and then the culture is frozen
in small aliquots at -70.degree. C..+-.10.degree. C. Master and
production seed stocks of the ETEC strain are prepared under
current good manufacturing practices (GMP). At about 18 to about 22
hours before the ETEC challenge, the required orally inocula are
obtained by thawing a single frozen vial and inoculating multiple
agar plates for overnight culture. The bacteria are harvested,
suspended in sterile saline and read at 600 nm in a
spectrophotometer. The concentration of cells is adjusted to a
stock solution of about 10.sup.10 to about 10.sup.9 cfu/ml. The
bacterial suspension may be diluted in saline. One (1) ml of the
bacterial suspension is administered to each subject.
[0057] The number of viable cfu (colony-forming units) administered
to a human subject may be determined retrospectively by serially
diluting, plating, and averaging the inoculum given to each subject
(pre- and post-challenge). Each dilution is done in triplicate. One
(1) ml of a bacterial suspension containing the target number of
cfu is mixed with 30 ml of sodium bicarbonate, 2 g of NaHCO.sub.3
per 150 ml of sterile, deionized water. This mixture is ingested by
the subject about 2 minutes after ingestion of 120 ml of the sodium
bicarbonate solution. At least 90 minutes before and after the
challenge, each subject should fast.
[0058] Subjects used for this assay should have a medical
examination and be determined to be in good health. Subjects should
have regular bowel habits, at least three stools (of normal
consistency) per week and two or fewer stools per day, without
frequent use of laxatives or antidiarrheal agents. Persons who were
previously exposed or possibly exposed to ETEC or cholera bacteria,
are allergic to quinolones, or have any significant
gastrointestinal abnormality should be excluded as well as pregnant
females, those being human immunodeficiency virus, hepatitis B
surface antigen or hepatitis C virus positive, those being treated
with antibiotics, theophylline, iron, zinc, histamine, H-2 receptor
antagonist or proton pump inhibitors, or those having febrile
illness within 48 hours prior to admission.
[0059] A double blind study may be conducted over a given period of
time, such as two weeks. Following ETEC challenge, each subject is
assessed about 3 to about 4 times daily for reactions to the
challenge. Symptoms are graded as mild (no limitation in activity,
no intervention needed), moderate (mild-to moderate limitation in
activity; no or minimal treatment and/or intervention needed), or
severe (unable or difficult to perform daily activities and
treatment and/or intervention needed). Vital signs are recorded at
least three times a day and fever is defined as an oral temperature
of greater than about 100.5.degree. F. Each stool output is
collected, weighed, assessed for presence of blood and graded as
previously described. See Coster, T. S., et al. (1995) Lancet 345:
949-952, which is herein incorporated by reference.
[0060] Any diarrhea is further classified as mild, moderate, and
severe. Mild diarrhea is defined as two to three loose stools,
which did not exceed 400 g diarrheal stools pre 24 hours. Moderate
diarrhea is four to five loose stools with 401 to 799 g diarrheal
stools per 24 hours. Severe diarrhea is defined as six or more
loose stools with 800 g or higher weight of diarrheal stool per 24
hours, or two loose stools/day, regardless of stool weight, and
fever. Severe diarrhea must also meet at least two of the following
symptoms graded as severe, including headache, nausea, generalized
aches, abdominal cramps, gas, emesis, malaise, or fatigue.
Colonization is defined as a positive test for the ETEC challenge
strain in two stool cultures collected at least 24 hours
post-challenge.
[0061] Stool samples from each subject are weighed and streaked
directly onto MacConkey agar culture medium. Spread plates are
inoculated after taking about 0.1.+-.0.01 g of stool and making 100
fold serial dilutions in phosphate buffer solution. Each dilution
are plated onto MacConkey agar. Stool samples (or rectal swabs if
no stool sample is produced) collected between the 0700 and 1900
hours are plated immediately. Samples collected at other times are
suspended in buffered glycerol saline, refrigerated at 4.degree.
C., and streaked for colony isolation the following day.
Presumptive ETEC isolates are confirmed by colony blot, with
detection by antisera raised in New Zealand white rabbits specific
for the given strain antibody. Stool is considered negative for
ETEC if no E. coli are isolated or are negative by immunoblot in
about ten picked colonies.
[0062] After the collected stool is weighed, small amounts of stool
specimens are immediately stored at -70.degree. C. Fecal antibodies
are extracted by the Ahren's procedure. See Ahren, C., et al.
(1993) Vaccine 11: 929-934. Five grams of thawed stool is mixed
with 5.0 ml of solution containing STI (100 mg/ml) (Sigma, St.
Louis, Mo.), EDTA (0.05M), and PMSF (10 mM) (Sigma), dissolved in
PBS supplemented with 0.05% Tween 20 (Sigma). The mixture is left
to stand at the room temperature for 15 minutes with intermittent
shaking and then centrifuged at 20,000.times.g for 30 minutes.
Bovine serum albumin (final concentration, 0.1%: weight/volume)
(Sigma) and sodium azide (final concentration, 0.02%) (Sigma) are
added to the supernatant after the pellet is discarded. Aliquots of
the fecal extract supernatant are stored at -70.degree. C. until
assayed for total and specific IgA antibody contents by ELISA. See
Jertborn, M., et al. (1988) Vaccine 16: 255-260, Stoll, B. J., et
al. (1986) J. Infect. Dis. 153: 527-534; and Svennerholm, A. M., et
al. (1983) J. Infect. Dis. 147: 514-522, which are herein
incorporated by reference.
[0063] The total IgA content in the fecal extract samples may be
determined by a modified ELISA method by using a human colostral
IgA reference (Sigma) as a standard. Specimens with an IgA
concentration of less than about 10 .mu.g/ml should be excluded
from further analysis, since antibody titrations of specimens with
IgA concentration of less than about 10 .mu.g/ml give unreliable
results. Specific antibody levels in the fecal extracts are
determined using conventional ELISA methods known in the art. To
adjust for variations in the IgA content in the stool samples
collected from different persons and on different days, fecal
extract antibody titers are expressed as units per microgram and re
obtained by dividing the specific titer (in units per milliliter)
by the total IgA content (in micrograms per milliliter), and
multiplying by 10. About a 2-fold or greater increase in the titer
of specific IgA per total IgA content between pre- and
post-challenge specimens is considered to be a responder.
[0064] Exploratory data analyses of the ETEC strains and doses with
all measured parameters are performed at periods, including the
control baseline prior to ETEC challenge, post-challenge of ETEC to
initiation of antibiotic treatment, and initiation of antibiotic
treatment plus 48 hours. See SAS/STAT User's Guide, Version 6, 2:
1007-1069, SAS Inst. Inc., Cary, N.C. 27513. Some data are analyzed
by Fisher exact 2-tailed test at a 95% confidence level. A Student
t-test may also be applied in certain cases. Furthermore, clinical
symptoms expressed in the occurrence ratios (reported cases/all
infected volunteers) are compared between different and the same
sexes. SPSS Base 10 software (SPSS Inc., Chicago, Ill.) was used to
analyze the immunological data, while other data were analyzed by
the SAS programs.
[0065] Generally, control subjects (not administered a given RBC
preparation) should exhibit a mean onset time of diarrhea and
colonization at about 8 to about 60 hours. After oral challenge
with the given ETEC strain, the first detection of fecal ETEC and
shedding post-challenge in the control subjects should be about 3
to about 60 hours. Symptoms of ETEC infection include diarrhea,
headache, gurgling, abdominal cramps, nausea, vomiting, appetite
loss, fatigue, tenesmus, fever, emesis, malaise and
lightheadedness.
[0066] An effective RBC preparation will prevent, inhibit, or
reduce the symptoms associated a disease or infection caused by the
ETEC challenge strain.
[0067] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference therein to the same extent as though each were
individually so incorporated.
[0068] Having thus described exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
within disclosures are exemplary only and that various other
alternatives, adaptations, and modifications may be made within the
scope of the present invention. Accordingly, the present invention
is not limited to the specific embodiments as illustrated herein,
but is only limited by the following claims.
* * * * *