U.S. patent application number 14/171513 was filed with the patent office on 2014-05-29 for method for removal of toxins from mucosal membranes.
This patent application is currently assigned to POLYRIZON LTD.. The applicant listed for this patent is POLYRIZON LTD.. Invention is credited to Smadar COHEN, Tomer IZRAELI, Robert S. MARKS.
Application Number | 20140147452 14/171513 |
Document ID | / |
Family ID | 36570282 |
Filed Date | 2014-05-29 |
United States Patent
Application |
20140147452 |
Kind Code |
A1 |
IZRAELI; Tomer ; et
al. |
May 29, 2014 |
METHOD FOR REMOVAL OF TOXINS FROM MUCOSAL MEMBRANES
Abstract
The present invention provides novel mucoadhesive compounds
useful in the prevention of diseases and disorders of or which are
associated with the mucosal membrane.
Inventors: |
IZRAELI; Tomer; (Rishon
Lezion, IL) ; COHEN; Smadar; (Beer-Sheva, IL)
; MARKS; Robert S.; (Omer, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
POLYRIZON LTD. |
RISHON LEZION |
|
IL |
|
|
Assignee: |
POLYRIZON LTD.
RISHON LEZION
IL
|
Family ID: |
36570282 |
Appl. No.: |
14/171513 |
Filed: |
February 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11885340 |
Nov 1, 2007 |
8679484 |
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PCT/IL06/00291 |
Mar 2, 2006 |
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14171513 |
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60738973 |
Nov 23, 2005 |
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60657388 |
Mar 2, 2005 |
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Current U.S.
Class: |
424/178.1 ;
435/375; 514/54; 530/391.7; 536/3 |
Current CPC
Class: |
C08B 37/0084 20130101;
A61K 47/61 20170801; A61K 47/6883 20170801; A61P 31/00
20180101 |
Class at
Publication: |
424/178.1 ;
530/391.7; 536/3; 514/54; 435/375 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. A compound of the formula A-(B).sub.n: wherein A is a
mucoadhesive backbone, B is a ligand group being chemically
substituted to A and capable of interacting with a toxin, and n is
the number of ligand groups substituted to said A, being greater or
equal to 1.
2. The compound according to claim 1, wherein said mucoadhesive
backbone is a polymer.
3. The compound according to claim 2, wherein said polymer is
selected from the group consisting of anionic polymer, cationic
polymer, non-ionic polymer and thiolated polymer.
4. The compound according to claim 1, wherein said polymer is
alginate and said ligand group is selected from the group
consisting of a saturated carbon chain of at least two carbon
atoms, aromatic ring, crown ether, cryptate, amide, carboxylic
acid, ester, sulfonate, sulfoxide, sulfoneamide, porphyrin,
metalloporphyrin, bis(dimethylglyoximate), bis(acetylacetonato),
bis(8-quinolinato), bis(4-fluorosalicylaldeydato), N,N'-ethylenebis
salicylideneiminato, macromolecule, peptides, extracellular matrix
proteins and biologically active molecule.
5. The compound according to claim 4, wherein said ligand group is
a macromolecule or a biologically active molecule.
6. The compound according to claim 5, wherein said biologically
active molecule is an antibody.
7. The compound according to claim 1, wherein said toxin is
selected from the group consisting of pollen, dander, mold, dust,
gas, vapor, dust molecules from industrial, medicinal or other
environmental sources, chemical warfare agents, biological toxins,
viruses, bacteria, fungus, and biological warfare agents.
8. The compound according to claim 7, wherein said toxin is a
virus.
9. The compound according to claim 7, wherein said toxin is
pollen.
10. The compound according to claim 1, wherein A is alginate, B is
an antibody and n is between 1 and 10.
11. The compound according to claim 10, wherein n is between 1 and
5.
12. The compound according to claim 10, wherein n is 1, 2 or 3.
13. (canceled)
14. A composition comprising a compound according to claim 1.
15. The composition according to claim 14, being a pharmaceutical
or hygienic composition.
16. A method for toxin removal from a mucosal membrane or the
environment thereof, said method comprising contacting said mucosal
membrane with an effective amount of a composition comprising at
least one compound according to claim 1.
17. A method for the prevention of a disease or disorder of the
mucosal membrane, being associated with a toxin, said method
comprising contacting a mucosal membrane of a subject with an
effective amount of a composition comprising at least one compound
according to claim 1.
18. A method for shielding a mucosal membrane from contacting
toxins or being penetrated thereby or adsorbed thereto, comprising
contacting said mucosal membrane with an effective amount of a
composition comprising at least one compound according to claim
1.
19. The method according to claim 16, wherein said mucosal membrane
is selected from the group consisting of a membrane of the mouth,
gastrointestinal tract (GI tract), nose, nasal cavity, lungs,
larynx, trachea, pharynx, vagina, rectum and urethra.
20. The method according to claim 19, wherein said mucosal membrane
is related to the respiratory system.
21. The method according to claim 16, wherein said toxin is
selected from the group consisting of pollen, dander, mold, dust,
gas, vapor, dust molecules from industrial, medicinal or other
environmental sources, chemical warfare agents, biological toxins,
viruses, bacteria, fungus, and biological warfare agents.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method for removal of toxins
from mucosal membranes.
BACKGROUND OF THE INVENTION
[0002] The mucosal membrane or tissue is a mucus-secreting membrane
which lines all body cavities or passages that communicate with the
exterior. While bioadhesion refers to the ability of certain
synthetic and biological macromolecules and hydrocolloids to adhere
to biological tissues, mucoadhesive compounds are capable of
adhering to mucosal membranes (mucosal tissues) through a complex
phenomenon, which in part depends upon the properties of compound,
biological tissue, and the surrounding environment. Several factors
have been found to contribute to a compound's bioadhesive capacity:
the presence of functional groups able to form hydrogen bridges,
the presence and strength of anionic charges, sufficient elasticity
for the polymeric chains to interpenetrate the mucous layer, and
high molecular weight. For this reason, most mucoadhesive compounds
are polymeric.
[0003] Mucoadhesive drug delivery systems exploit the attraction
between the mucus and polymeric drug carrier. They provide
localization of the carriers within the specific site and prolonged
residence time of the delivery devices. These greatly enhance the
bioavailability of the drugs, especially in the case of peptide and
protein delivery. Such systems have been used in dentistry,
orthopedics, ophthalmology, and in surgical applications and
recently with the emergence of controlled release systems for local
release, such applications include also systems for release of
drugs in the buccal or nasal cavity, and for intestinal or rectal
administration.
[0004] Mucoadhesive vaginal formulations have also been disclosed
for example by Gavini et al (Mucoadhesive vaginal tablets as
veterinary delivery for the controlled release of an antimicrobial
drug, acriflavone: AAPS PharmaSci 2002; 3(3) article 20) and C.
Valenta (The use of mucoadhesive polymers in vaginal delivery:
Advances Drug delivery Reviews 2005: 57, 1692-1712). Nasal drug
delivery using mucoadhesive carrier has also been described (Nasal
mucoadhesive drug delivery: Background, applications, trends and
future perspectives: Ugwoke et al., Advanced Drug Delivery Reviews
2005; 57, 1640-1665).
[0005] US application no. 2005/0281775 discloses a method for
augmenting an epithelial mucosal harrier by contacting the barrier
with a topical composition which comprises a mucoadhesive polymer.
The method and compositions discloses are said of being useful in
improving mucosal barrier function by, for example, topical
application to an exposes or injured epithelial surface or by
coating a compromised mucosal barrier in inflammatory bowel
disease.
SUMMARY OF THE INVENTION
[0006] It has been found in accordance with the present invention
that mucoadhesive systems may be utilized not only for the delivery
of pharmaceuticals and other active agents into the mucosal
membranes or mucosal tissues, as shown extensively by existing
prior art, but also remove therefrom toxins which can cause
allergies and other deficiencies. The removal of such toxins from
the respiratory system, for examples, may be achieved by binding
one or more of said toxins to novel mucoadhesive ligand systems.
Such systems may thus find utility as therapeutics as well as
agents for hygienic and prophylactic purposes.
[0007] Thus, in one aspect, the present invention provides a
compound of the formula A-(B).sub.n:
[0008] wherein A is a mucoadhesive backbone, B is a ligand group
being chemically substituted to A and capable of interacting with a
toxin, and n is the number of ligand groups chemically substituted
to said A, being greater or equal to 1.
[0009] The "mucoadhesive backbone", A, is the matrix, chain,
polymer or other chemical entity to which the ligand groups, B, are
substituted. Backbone A may be biodegradable or non-biodegradable
in nature and may be selected based on several parameters such as
their intended use and targeted mucosal membrane. Such backbone may
be organic, namely substantively composed of carbon atoms;
inorganic such as a silica backbone; or a mixture of both. The
mucoadhesive backbone may be selected from the group of natural or
synthetic (or combination thereof) polymers, metalopolymers, cross
linked polymers, polysaccharides, organic-inorganic polymers, and
peptides. The backbone may additionally be charged, i.e. may be
presented as a salt.
[0010] The polymeric mucoadhesive backbone may be an anionic
polymer such as poly(acrylic acid), carrageenan, carbopol,
polycarbophil, poly(methacryl acid), alginate,
carboxymethylcellulose, sodium hyaluronate; cationic polymer such
as chitosan; non-ionic polymer such as hydroxymethylcellulose,
hydroxypropylcellulose, polyvinylpirrolidone, polyethyleneglycol;
or thiolated polymer such as cysteine conjugates of poly(acrylic
acid), polycarbophil and sodium carboxymethylcellulose,
2-iminothiolane chitosan. Additional examples of mucoadhesive
polymers are tamarind seed polysaccharide, gelatin, gliadin,
pectin, poly-N-vinylpyrrolidone, xanthan gum, and 2-acrylamido
2-methylpropane-sulfonic acid.
[0011] The polymers may be homopolymer, copolymer, terpolymer, or
interpolymer. These polymers are preferably ionic, and are selected
from polycarboxylic acids, polysulfonic acids, or salts thereof.
Polysulfonic acids include sulfoethylmethacrylate, sulfopropyl
methacrylate, sulfopropyl acrylate, N,N-dimethyl-N-methacryl
oxyethyl-N-(3-sulfopropyl)ammonium betaine, itaconic
acid-bis(1-propyl sulfonizacid-3)ester dipotassium salt and
methacrylic acid amidopropyl-dimethyl ammonium sulfobetaine.
[0012] Examples of acrylic acid or polyacrylic acid polymers
include those having monomers of methyl acrylate, ethyl acrylate,
propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl
acrylate, methyl methacrylate, methyl ethacrylate, ethyl
methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate,
isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate,
hexyl acrylate, n-hexyl methacrylate, and the like. Higher alkyl
acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl
acrylate, stearyl acrylate, behenyl acrylate and melissyl acrylate.
Mixtures of two or three or more long chain acrylic esters may be
successfully polymerized with one of the carboxylic monomers.
[0013] Specifically preferred mucoadhesive polymers are sodium
alginate, Chitosan, Chitosan modified with thioglycolic acid (TGA)
onto the primary amino groups, Chitosan-4-thio-butyl
amidin-conjugates (chitosan TBA), Hyaluronic acid and derivatives,
Pectin and traganth, Starch, Sulfated polysachcharides such as
heparin, dextran sulfate, sulfated cyclodextrins, Carrageenan,
Gelatin, Sodium carboxy methyl cellulose (CMC) and derivatives,
methyl cellulose (MC), Synthetic polymers such as poly(acrylic
acid) (PAA) and derivatives, hydroxypropyl methylcellulose and
poly(methylacrylate) derivatives, and thiolated polymers.
[0014] In one preferred embodiment of the invention, the
mucoadhesive backbone is a polymer, most preferably alginate. Thus,
there are provided the following compounds of the general formula
A-(B).sub.n wherein A is alginate, n is an antibody and n is 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10. In one preferred embodiment, B is an
antibody capable of binding to a virus selected from adenovirus,
rhinovirus, Rift Valley virus, Ebola virus, and influenza
virus.
[0015] There are thus provided the following conjugates:
[0016] A conjugate of alginate with an antibody for an
adenovirus,
[0017] A conjugate of alginate with an antibody for a
rhinovirus,
[0018] A conjugate of alginate with an antibody for the Rift Valley
virus,
[0019] A conjugate of alginate with an antibody for the Ebola
virus,
[0020] A conjugate of alginate with an antibody for an influenza
virus.
[0021] Preferably, the ratio of alginate to the antibody is 1 to 1,
1 to 2, or 1 to 3, respectively. The ratio of alginate to the
antibody may also be 1 alginate to at least 4 antibodies.
[0022] The compound of the general formula A-(B).sub.n, also termed
the "mucoadhesive ligand" may be one having a mucoadhesive backbone
with at least one ligand group, B, emerging therefrom in an
arrangement which may be symmetric or asymmetric and may comprise a
single type ligand, i.e. being selective towards a single toxin, or
a multi-type ligand, i.e. being selective to a range of toxins.
Non-limiting arrangements of the compounds of general formula
A-(B).sub.n are shown in FIGS. 1A-E.
[0023] The "ligand group", B, refer to an ion, a functional group
or a side group, which is chemically substituted (namely, having at
least one bond therewith, such bond may be covalent, ionic,
electrostatic, hydrogen bond, van der Waals interaction, London
force or any combination thereof) onto the mucoadhesive backbone,
A, or embedded therein, by means of physical attraction and which
is capable of interacting with a toxin to which it is exposed. This
interaction, e.g. by formation of a bond (namely, having at least
one bond therewith, such bond may be covalent, ionic,
electrostatic, hydrogen bond, van der Waals interaction, London
force or any combination thereof) may be such that allows strong
enough interaction with said toxin so as not to allow release of
the toxin, once captured, back into the mucosal membrane. Such
interaction may through any type of chemical or physical
interaction and, for example, may be through co-ordinate covalent
bond and may be polydentate or bidentate in nature.
[0024] The ligand group may also be a reactive group that undergoes
a chemical transformation when in contact with the toxin. For
example, the ligand may be an unsaturated carbon chain capable of
undergoing reduction e.g., halogenation in exposure to toxins (e.g.
halogens) or may be a metal ion capable of undergoing a redox-type
transformation upon exposure to a variety of chemical toxins. The
ligand may also be a liposome or a micro- or nanoparticle capable
of enclosing a toxin.
[0025] The ligand group may also be a so-called proligand which is
susceptible to chemical transformation in the targeted organ,
thereby transforming into a reactive entity only when arriving at
the target membrane. For example, a proligand may be an esterified
acidic chain which undergoes acidic saponification at the target
membrane, reverting into the active acidic chain and binding the
toxins which it encounters.
[0026] The ligand functional group may for example be a unsaturated
carbon, chain of at least two carbon atoms, aromatic rings, crown
ethers, cryptates, amides, carboxylic acids, esters, sulfonates,
sulfoxides, sulfoneamides, porphyrins, metalloporphyrins,
bis(dimethylglyoximate), bis(acetylacetonato), bis(8-quinolinato),
bis(4-fluorosalicylaldeydato), N,N'-ethylenebis salicylideneiminato
and the like. The ligand may also be a macromolecule or a
biologically active molecule such as a soluble receptor or a
receptor antagonist, protein or an antibody which is capable of
interacting with a toxin such as a virus.
[0027] in one preferred embodiment, B is an antibody capable of
interacting with a virus (the toxin) selected from Variola major
and other pox viruses; Arenaviruses such as Junin virus, Machupo
virus, Guarnarito virus, ymphocytic choriomeningitis virus, Lassa
virus; Bunyaviruses such as Hantaviruses, Rift Valley fever virus;
Flaviruses such as Dengue fever viruses; Filoviruses such as Ebola
virus, Marburg virus; Eastern equine encephalitis virus, Western
equine encephalitis virus, Venezuelan equine encephalitis virus, La
Crosse virus, Japanese encephalitis virus, Kyasanur forest virus,
California encephalitis viruses; food and waterborne Pathogens such
as Caliciviruses; Hepatitis A virus; Nipah virus; Yellow fever
virus; Influenza viruses; Rabies virus and other Hantaviruses.
[0028] In another embodiment, the ligand group B is a ligand
capable of trapping pollen particles, chemically, i.e. by binding
to one or more components which cover the outer surface of the
pollen, or physically, i.e. by engulfing the pollen. Such ligand
groups may be selected from amino acids such as phenylalanine,
leucin, valine, isoleucine, arginine, histidine, lysine, methionine
and others which may be bonded to the polymer A through the C- or
N-terminal of the amino acid group; short peptides not exceeding
about 10 kD in size, which may be connected to the polymer A
through the C- or N-terminal; sugars such as fructose, glucose or
sucrose which may be connected to the polymer A through one or more
of the alcohol groups or oligo or polysaccharides. Physical
trapping of the pollen may take place, as will be shown below, for
example, by the mucoadhesive polymer itself which interacts with
the pollen by engulfing it.
[0029] The mucoadhesive backbone and the ligand as a whole may be
obtained from mammalian tissues, such as hyaluronic acid, dermatan
sulfate and chondroitin sulfate; from the exoskeleton of
crustaceans, such as chitin that further deacetylated to obtain
chitosan; from vegetables and plants, such as starch and starch
derivatives; from aquaculture sources such as alginate, or they may
be synthetically modified in order to obtain the desired
ligand-toxin specificity or interaction.
[0030] The term "toxin" as used herein refers to a substance which
can cause a long-term or short-term, local or systemic toxicity to
the mucosal membrane, the organ said membrane is associated
therewith or to the whole animal body (human or non-human). Such
toxicity may for example be an allergic reaction or any other
long-term or short-term non-allergic reaction such as viral
infection, bacterial infection, fungal infection, poisoning by
biotoxins and others.
[0031] The toxins may enter the body of the subject through the
opening of a body cavity, e.g. mouth, nose by inhalation or by
contamination by other air-borne particulates or by other
particulate contaminants. The origin of the toxins may also be the
body itself. For example, toxins which are expelled from the body
through breathing may be captured by the mucoadhesive compounds,
thereby minimizing exposure of the subject's environment to said
toxins.
[0032] Examples of particulate toxins, without being limited
thereto are pollen, dander, mold, spores of various origins and
dust. Examples of chemical toxins are gas, vapor or dust molecules
from industrial, medicinal or other environmental sources such as
halogen gases, e.g., bromine, chlorine gases, chlorofluorocarbon
(CFCs), NOx, SOx, CO, nicotine, nicotine by-products, smoking
by-products such as acetaldehyde, formaldehyde and others, metal
ions, charged or uncharged molecules such as silicon, asbestos,
chemical warfare agents, and others. Also included are biological
toxins (biotoxins) such as animal or plant toxins, viruses,
bacteria, fungus, and biological warfare agents.
[0033] Non-limiting examples of the viral toxins are Adenoviruses,
Arboviruses, Arenaviruses, Encephalitis, Orthomyxoviruses,
Papillomaviruses, Paramyxoviruses, Picornaviruses, Poxviruses,
Retroviruses, Rhabdovirus, and Rhinoviruses. Specific examples are
Variola major and other pox viruses; Arenaviruses such as Junin
virus, Machupo virus, Guanarito virus, ymphocytic choriomeningitis
virus, Lassa virus; Bunyaviruses such as Hantaviruses, Rift Valley
fever virus; Flaviruses such as Dengue fever viruses; Filoviruses
such as Ebola virus, Marburg virus; Viral encephalitis such as West
Nile virus, Eastern equine encephalitis virus, Western equine
encephalitis virus, Venezuelan equine encephalitis virus, La Crosse
virus, Japanese encephalitis virus, Kyasanur forest virus,
California encephalitis viruses; Food and Waterborne Pathogens such
as Caliciviruses; Hepatitis A virus; Nipah virus; Tickborne
hemorrhagic fever viruses such as Crimean-Congo hemorrhagic fever
virus; Tickborne encephalitis viruses; Yellow fever virus;
Influenza and avian influenza viruses; Rhinoviruses. Rabies virus
and other Hantaviruses.
[0034] Non-limiting examples of the bacterial toxins are Bacillus
anthracis; Clostridium botulinum; Francisella tularensis; Yersinia
pestis; Burkholderia pseudomallei; Burkholderia mallei; Clostridium
perfringens; Coxiella burnetii; Brucella melitensis, abortus, suis,
and canis; Staphylococcus aureus; Rickettsia prowazekii; Chlamydia
psittaci; Food and Waterborne Pathogens such as Escherichia coli
O157:H7, Vibrio cholera; Salmonella species, Shigella species,
Listeria monocytogenes, Campylobacter jejuni, Yersinia
enterocolitica; Mycobacterium tuberculosis; and other
Rickettsia.
[0035] Non-limiting examples of chemical warfare agents are the
persistent or non-persistent nerve agents such as tabun, sarin,
soman, GF, and VX (methylphosphonothioic acid); blister agents such
as sulfur mustard, nitrogen mustard, Lewisite, and phosgene
oximine; choking agents such as phosgene, diphosgene, chlorine and
chloropicrin. Also included are lacrimators such as
chlorobenzylidenemalononitrile, chloroacetophenone, and
nitrochloromethane.
[0036] Non-limiting examples of the biological warfare agents are
anthrax, Botulinum toxins, Brucellosis, cholera, Clostridium
perfringens toxins, Congo-Crimean hemorrhagic toxins, Ebola,
Melioidosis, plague, Q fever, richi, Rift valley toxin, Saxitoxin,
smallpox, Staphylococcal enterotoxin B, Trichothecene mycotoxins,
tularemia, and Venezuelan equine encephalitis.
[0037] Other toxins may be food and waterborne pathogens such as
Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia,
Entamoeba histolytica, Toxoplasma gondii, Microsporidia, and plant
pathogens such as Ricinus communis Castor bean.
[0038] Preferably, the toxins are pollen, dander, mold, and dust,
variola major, Marburg toxin, Rift Valley fever virus, Ebola virus,
Clostridium botulinum toxin, Anthrax Bacillus anthracis, RSV,
Rhinoviruses, spores of Aspergillus sp., yeast of Cryptococcus
neoformans, arthro spores such as Coccidiodes immitis and
Influenza.
[0039] Also encompassed are toxins which are unique to hospitals
which are capable of causing the so-called hospital associated
infections (HAI). One example of such a toxin is the
methicillin-resistant Staphylococcus aureus (MRSA). MRSA is the
most infamous hospital pathogen, with levels of resistance
increasing from 3% in 1992 to 43% in 2002 in England and Wales (HPA
2003). However, HAI caused by other micro-organisms such as
vancomycin-resistant enterococci (VRE), Clostridium difficile,
Acinetobacter baumannii and multi-drug resistant (MDR)
Acinetobacter sp. are becoming increasingly prevalent and are thus
within the scope of the term toxin.
[0040] The presence of or contact with such toxins may directly or
indirectly result in the occurrence of local or systemic disorders
or diseases which are known to be associated with the specific
toxin (pathogen) or the family with which it is associated. Such
diseases or disorders may be diseases of the respiratory system,
e.g. asthma, adult and infant respiratory distress syndrome,
suffocation, atelectasis, bronchiectasis, carbon monoxide
poisoning, cancer, chronic obstructive pulmonary disease, cystic
fibrosis, fluid accumulation in the alveoli, pneumonia, sinusitis,
tuberculosis, and others; viral injections, Smallpox, Argentine
hemorrhagic fever, Bolivian hemorrhagic fever, Venezuelan
hemorrhagic fever, Lymphocytic choriomeningitis, Lassa fever,
Hantavirus pulmonary syndrome, Rift Valley fever, Dengue fever,
Ebola hemorrhagic fever, Marburg hemorrhagic fever, Eastern equine
encephalitis, Western equine encephalitis, Venezuelan equine
encephalitis, La Crosse encephalitis, Japanese encephalitis,
Kyasanur forest disease, California encephalitis, Gastmenteritis,
Hepatitis A, Nipah virus encephalitis, Crimean-Congo hemorrhagic
fever, Yellow fever, Flu, Rabies, and others; bacterial infection;
e.g. Anthrax, Botulism, Tularemia, Plague, Melioidosis, Glanders,
Epsilon toxin, Q fever, Brucellosis/Undulant fever, Enterotoxin B,
Epidemic typhus, Psittacosis, Diarrheagenic E. coli, Cholera,
venereal disease, Salmonellosis, Shigellosis, Listeriosis,
Campylobacteriosis, Yersiniosis, Multidrug-resistant Tuberculosis,
and Rickettsial diseases; and diseases and disorders associated
with food and waterborne toxins such as Cryptosporidiosis,
Cyclosporiasis, Giardiasis, Amebiasis, Toxoplasmosis
Microsporidiosis, and others.
[0041] In a further feature of the present aspect, there is
provided a further mucoadhesive ligand having the formula
(D).sub.m-A-(C).sub.n, wherein. A is a mucoadhesive polymer, C is a
first ligand group being chemically substituted to A and capable of
interacting with a toxin, D is a second ligand group being
chemically substituted to A and reversibly chelated to an agent
capable of being released from said second ligand group, n and m
each independently integers greater than 1. Non-limiting examples
of the compounds of general formula (D).sub.m-A-(C).sub.n are shown
in FIGS. 2A-C.
[0042] Upon application of a compound of the general formula
(D).sub.m-A-(C).sub.n, or a composition comprising thereof, to the
mucosal membrane, the second ligand group, D, may release its
chelated agent to the mucosal membrane or to the environment
thereof, and the free ligand group, C, may act to remove therefrom
any toxin for which it is designed.
[0043] The compound of the general formula (D).sub.m-A-(C).sub.n,
may be administered in a fully loaded form, namely having a
substantial number of said D groups bound to an agent to be
released into the mucosal membrane. Preferably, the compound of the
general formula (D).sub.m-A-(C).sub.n, is used with a maximal
number of said ligands, D, being bound to an agent to be
released.
[0044] The compound of the general formula (D).sub.m-A-(C).sub.n,
may also be administered in a partially-bound form, namely having
only a portion of said D groups bound to an agent to be released
into the mucosal membrane. The term "partially" or "semi" does not
stand to mean any specific number or percentage of bound or loaded
ligands from the total number of ligand groups attached to the
backbone of any one mucoadhesive backbone. The following
non-limiting exemplary ligands are considered as semi-loaded
ligands: a) a polymer A having 100 ligand groups D of which one is
loaded; b) a polymer A having 2 ligand groups D of which one is
loaded; c) a polymer having 50 ligand groups D of which 48 are
loaded, etc.
[0045] Such partially-loaded mucoadhesive ligands may be used in
the method disclosed herein for removal of toxins from mucosal
membranes and have the added benefit of being able to replenish or
deliver selected substances to the membrane or environment from
which toxins are to be removed. Examples of substances which may be
delivered to the membrane are: metal ions such as sodium,
magnesium, calcium; sugars, drugs; enzymes; growth-stimulating
agents; antidepressant agents; antibiotics; antiviral agents;
antiprotozoal agents; vitamins; and other agents which typically
form part of the mucosal membrane.
[0046] In one specific embodiment, the release of the substance
from the loaded ligand may be simultaneous with the capture of the
toxin by the free ligand C or may be independent thereof. In
another specific embodiment, said release is dependent on binding
by the free ligand group C by a toxin.
[0047] The ligand groups designated herein by B, C and D may be
arranged on the polymer A backbone randomly or selectively as
decided by the person synthesizing the compounds of the invention.
Exemplary, non-limiting arrangements are shown in FIGS. 1A, B, C, E
and 2A, B and C. In these figures, A and B are as defined
hereinabove. The entities labeled L are the chelation cavity or
active site of the ligand groups B to which the toxin attaches.
[0048] All compounds of the present invention may be biodegradable,
non-biodegradable or partially biodegradable in nature and may be
selected based on several parameters such as their intended use and
targeted mucosal membrane.
[0049] The compounds of the invention may be used for the
preparation of a therapeutic, prophylactic or hygienic
compositions, for example for the prevention of a disease or
disorder associated with the exposure to an opportunistic toxin
entering the environment surrounding said membrane and which
contact with said membrane may bring about a direct or in direct
toxicity (such as allergies, poisoning, infections etc). In one
embodiment, said compounds are used for the preparation of a
composition suitable for therapeutic purposes such as to prevent or
reduce the concentration of a certain toxin in a mucosal membrane.
In another case, the compositions are used for hygienic
purposes.
[0050] In a further embodiment, the compositions or the present
invention may further comprise one or more of a variety of agents
such as for example pH adjusters, carriers, excipients, diluents,
antibiotics, antioxidants, vehicles such as starch,
microcrystalline cellulose, lactose, sorbitol, or mannitol;
lubricants such as magnesium stearate, glycerol behenate, talc,
hydrogenated ricin oil or waxes; flow agents such as colloidal
silica; aromas, flavouring agents, sugaring or sweetening agents
and in general any substances capable of improving taste, odor or
appearance of the composition, and one or more pharmaceutical
agents such as glucocorticoids, dexamethasone, dexamethasone salts,
isothiozolones, anticoagulants, heparin, hirudin, peptides such as
oligopeptides and polpeptides, oligopeptides, antimitotic agents,
angiopeptin, polynucleotides, and oligonucleotides, sulfyhdryls,
hydroxamic acids, oral compositions including bioadhesive syrups
and gels, mouth wash, cough syrups and oral gels for mouth
sores.
[0051] The composition to be administered are typically
administered directly, namely in case of the respiratory system and
the GI tract-intranasally or per oral as a liquid or sprayed into
the oral cavity or on the back of the throat; in case of the vagina
as suppositories, vaginal rings, tablets, capsules, powders,
granules, or microgranules. The composition may also be delivered
topically, for example, to the oral cavity or to the vagina as a
dry powder which encores to the mucosal membrane after having
absorbed water therefrom or after having been absorbed therein. The
frequency of application may vary considerably, for example from
once per day to several times per day, or once or more in several
days, depending for example on the condition of the subject in need
of such composition. The composition may also be administered by
any other regimen as known to a person skilled in the art.
[0052] In another embodiment of the present invention, the
composition comprising of the mucoadhesive ligand may further
comprise or be administered in conjunction with enzyme inhibiting
agents such as reverse transcriptase inhibitors, protease
inhibitors, angiotensin converting enzymes, 5-alpha-reductase, and
the like. Typical agents include peptide and non-peptide agents
including finasteride, lisinopril, saquinavir, quinapril, ramipril,
indinavir, ritonavir, nelfinavir, zalcitabine, zidovudine,
allophenyinorstatine, kynostatin, delaviridine, bis-tetrahydrofuran
ligands, and didanosine.
[0053] The pharmaceutical composition of the present invention may
be formulated in the form of suppositories, tablets, films,
patches, and gels for oral, buccal, nasal, ocular, and vaginal
routes; as well as spray formulations, drops, dry powder,
suspensions and the like. The composition may additionally be
formulated as microparticles or nanoparticles.
[0054] In another aspect of the present invention, there is
provided a method for toxin removal from a mucosal membrane or the
environment thereof, said method comprising contacting said mucosal
membrane with an effective amount of a composition comprising at
least one compound of the general formula A-(B).sub.n, as defined
herein.
[0055] In one embodiment, the mucosal membrane is ex-vivo, namely
it is not part of a living animal body but rather one which is used
for example for laboratory, research or other medicinal
applications. Such membrane may be in a laboratory apparatus or a
Petri dish or in any other form as may be appropriate. In another
embodiment, said mucosal membrane is in a living animal such as
human or non-human animals. Preferably, it is part of a human.
[0056] The invention further provides a method for the prevention
of a disease or disorder of the mucosal membrane, said disease or
disorder being associated with a toxin, said method comprising
contacting a mucosal membrane of a subject with an effective amount
of a composition comprising at least one compound of the general
formula A-(B).sub.n.
[0057] The invention still further provides a method for the
prevention of a disease or disorder, comprising contacting a
mucosal membrane of a subject with an effective amount of a
composition comprising at least one compound of the general formula
A-(B).sub.n.
[0058] The invention in yet another of its aspects provides a
method for shielding a mucosal membrane from contacting toxins or
being penetrated thereby or adsorbed thereto, comprising contacting
said mucosal membrane with an effective amount of a composition
comprising at least one compound of the general formula
A-(B).sub.n.
[0059] Preferably, the mucosal membrane is selected from a membrane
of the upper or lower respiratory system, more preferably to the
mouth, gastrointestinal tract (GI tract), nose, nasal cavity,
larynx, trachea, pharynx, vagina, rectum or urethra. In some
embodiments, more than one membrane may be treated at one time,
with an identical or different composition. In one example, both
the nasal and GI tract are treated with a composition comprising a
conjugate of alginate and an antibody for the Rift Valley virus. In
another example, the nasal mucosa is treated with a conjugate of
alginate and an antibody for the Rift Valley virus and the vagina
is treated with a different conjugate or a composition comprising
same.
[0060] Most preferably, the mucosal membrane is related to the
respiratory system and specifically to the upper respiratory
system.
[0061] The administration of the compositions of the invention may
be by any known method as disclosed herein. Preferably, the
compositions are administered directly to the mucosal membrane by
topical administration or by direct contact therewith.
[0062] The term "contacting" as used herein is synonymous to
"administration". The terms or any lingual variation thereof refer
typically to topical administration of the composition of the
invention directly to the mucosal membrane, utilizing any type of
application known to a person skilled in the art. The composition
to be administered is typically applied directly by touching the
membrane with the composition. For example, in the case of the
respiratory system and the GI tract, the composition may be
administered intranasally or per oral as a liquid or sprayed into
the oral cavity or on the back of the throat. In case of the
vagina, the composition may be administered as suppositories,
vaginal rings, tablets, capsules, powders, granules, or
microgranules. The composition may be delivered topically with the
frequency of application ranging from once per day to several times
per day, or once or more in several days, depending on the
condition of the subject in need of such composition. The
composition may also be administered by any other regimen e.g.
systemic administration. The composition of the invention may be
administered in a dosage form or in separate dosages.
[0063] The invention further provides the use of mucoadhesive
polymers such as alginate for the preparation of a composition for
toxin removal from a mucosal membrane or its environment, such as
the skin surrounding the body cavity. The mucosal membrane
composition which is administered to the surrounding of the mucosa
may be applied in any known method, e.g. as a skin ointment.
[0064] Mucoadhesive polymers which are capable of entrapping a
toxin as defined herein may be used also for the treatment of
disease and disorders of the mucosal membrane or disease and
disorders which are associated with the penetration of such toxins
through the mucosal membranes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] In order to understand the invention and to see how it may
be carried out in practice, a preferred embodiment will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0066] FIGS. 1A-E are schematic representations of various
exemplary mucoadhesive ligands (A=mucoadhesive backbone; B=ligand
or chelating group; and L=chelation cavity).
[0067] FIGS. 2A-C are schematic representations of free, semi- or
fully loaded mucoadhesive ligands (A=mucoadhesive backbone;
B=ligand or chelating group; and L=chelation cavity).
[0068] FIG. 3 depicts the synthetic strategy to couple alginate
with different antibody to trap pathogenic biological entities.
[0069] FIG. 4 depicts the synthetic strategy for producing an
alginate conjugate with a ligand protein.
[0070] FIG. 5 shows the direct fluorescent assay for the
quantification of the alginate modification.
[0071] FIG. 6 shows the indirect assay for the quantification of
the target virus binding capacity using fluorescently labeled
virus.
[0072] FIG. 7 depicts the ELISA assay for the quantification of the
target virus binding capacity.
[0073] FIGS. 8A-B depict the kinetic study for the binding of the
target molecule. (A) Immobilization of the conjugate of alginate
with the ligand on the surface of the avidin coated slide, (B)
binding kinetics using the fluorescent depletion method.
[0074] FIGS. 9A-B depict the kinetic study of the retention of the
target molecule. (A) immobilization of the conjugate of alginate
with ligand on the surface of the avidin coated glass or gold
coated slide, (B) binding kinetics study utilizing fluorescence the
enrichment method.
[0075] FIG. 10 shows a scanning electron microscope (SEM) picture
of free (unsubstituted) alginate entrapping Easter Lily pollen.
DETAILED DESCRIPTION OF THE INVENTION
[0076] The mucosal membrane may be in the mouth, gastrointestinal
tract (GI tract), nose, nasal cavity, larynx, trachea, pharynx,
vagina, rectum or urethra and may absorb or be in direct contact
with toxins which penetrate it or reside in its environment such as
the volume of air above it or in the liquid which surrounds it. The
ligands of the present invention are capable of bonding
irreversibly (or substantially irreversibly for the period of their
residence in the subject's body) to such toxins and assist in their
removal from the treatable environment, e.g. the respiratory
system. Removal of such toxins from the treated environment may
allow in the prevention of conditions such as those disclosed
hereinbelow or in the treatment of an existing condition by
reducing the concentration of a toxin in the treated organ, i.e.
the respiratory system, nasal cavity, vagina and the like.
[0077] The term "respiratory system" refers to the complete
respiratory system, but preferably relates to the upper respiratory
system which includes the nasal cavity, the pharynx, trachea and
larynx.
[0078] The nasal cavity is divided laterally into two mucus-coated
passages, one from each nostril, by a midline septum containing
hyaline cartilage. From both lateral walls of the septum into the
two cavities are extending three curved plates of bone, the
superior, middle, and inferior conchae (turbinate bones) which are
covered by mucous membrane. The inferior and middle conchae are
covered by respiratory epithelium and the superior conchae is
covered by olfactory epithelium. The conchae function by increasing
the surface area containing respiratory epithelium and by creating
turbulence thus resulting in increase contact of the air and the
airborne toxins with the epithelium allowing the mucus secreted by
the respiratory epithelium to trap these toxins and allowing
humidification and warming of the air.
[0079] The pharynx connects the nasal and oral cavities to the
larynx and esophagus and acts as a passageway for air and food. The
parts of the pharynx with food contact (oropharynx and laryngeal
pharynx) are lined by nonkeratinizing-stratified squamous
epithelium. The part of the pharynx above the soft palate, the
nasopharynx, is lined by pseudo-stratified ciliated columnar
epithelium with goblet cells. The connective tissue of the pharynx
is fibroelastic surrounded by striated muscle of the pharyngeal
muscles. The pharyngeal tonsil, located in the midline of the
posterior wall of the nasopharynx, is made up of multiple confluent
lymphoid nodules intimately associated with the pseudo-stratified
columnar epithelium.
[0080] The larynx contains hyaline and elastic cartilage forming a
complex muscular (skeletal) architecture that maintains passage,
prevents swallowed food or liquid from entering the trachea in a
valve-like manner, and controllably produces sound. The epiglottis
with its central elastic cartilage plate extends into the pharynx
has stratified squamous epithelium on its anterior portion
(continuous with posterior surface of tongue). The posterior
surface has a ciliated, pseudo-stratified columnar epithelium
containing seromucous glands. The mucosa below the epiglottis has
two pairs of folds forming the vocal cords. The upper pair
represents the false (ventricular) vocal cords typically covered by
respiratory epithelium with serous glands within the lamina
propria. The lower pair constitutes the true vocal cords that are
covered by a stratified squamous epithelium devoid of glands.
[0081] Trachea morphology is best characterized by the large
C-shaped rings of hyaline cartilage, like 16-20 stacked horseshoes,
which maintain patency of this passageway to the lungs. This
flexible, semi-rigid tubular structure terminates where it
bifurcates into the two main bronchi. The posterior open portion of
the cartilage rings is bridged by fibroelastic ligament and smooth
(trachealis) muscle. Contraction of this muscle permits some
constriction of the tracheal lumen whereas the ligament prevents
dilation by overdistension. The luminal surface is typical
respiratory epithelium, containing columnar ciliated cells, mucus
secreting goblet cells, undifferentiated (stem) basal cells, and
submucosal seromucous glands.
[0082] The term "mucoadhesive" as used herein refers to a
phenomenon where a substance (of any source, i.e., natural,
synthetic or a combination thereof), when applied to a mucosal
epithelium, adheres to the mucosal layer for a period of time
sufficient to bind to airborne or other toxins, as discussed
herein. The mucoadhesive substances, which are typically polymers,
may be natural or synthetic and may be neutral or charged (anionic
or cationic).
[0083] Such substances adhering to the mucosal layer may have
varying retentive qualities. The preferred mucoadhesive substances
are those that are additionally "mucoretentive". The retentive
properties of such substances refer to the substance's, or
composition containing thereof, degree of resistance to washing and
dissolving forces of fluids in the respiratory system.
[0084] The mucoadhesive properties of the substances used and their
capability in binding and removing toxins which approach the
mucosal membrane may be assessed by comparison to control
compositions that do not contain the mucoadhesive substance.
[0085] Generally speaking, the compounds of the invention are
bifunctional, namely have: (1) a polymer as the mucoadhesive
backbone, which promotes adhesiveness and retention of the
mucoadhesive ligand to the mucus membrane, and (2) at least one
functional group, a ligand or a chelator, which is capable of
bonding, chemically or physically with the toxin or a ligand or
chelator already loaded with a certain agent which may be released
after application to the mucus membrane for example for
replenishment of said agent.
[0086] Also encompassed within the scope of the invention are those
ligand molecules which although lacking a distinctive backbone,
still maintain mucoadhesiveness and chelator properties. Such may
for example be liposomes.
[0087] Binding of the toxin may also be achieved through the
mucoadhesive backbone and not via the chelating functional group.
In such cases, in the general formula A-(B).sub.n the mucoadhesive
substance itself may act as the ligand system and the ligand group
may need not be present (n=0). Such a binding power is shown in the
SEM picture of FIG. 10, which depicts free alginate after having
contacted a concentration of Easter lily pollen. The entrapment of
the pollen within the polymer is exhibited.
[0088] Additionally, the mucoadhesive ligands may be constructed as
a nanoparticles or microspheres. Suitable mucoadhesive microspheres
are for example carboxyvinyl mucoadhesive microspheres, chitosan
microspheres, Eudragit floating microspheres, and cholestyramine
microparticles.
[0089] The ligand groups may be such groups that are part of the
mucoadhesive backbone or may be selectively attached thereto using
chemical transformations as known to a person skilled in the art of
organic or inorganic synthesis (see for example: Organic Synthesis,
John Wiley & Sons, New-Jersey, 2003; Sandler S R., Kayo W.,
Organic Functional Group Transformations, Academic Press, 1971;
Fieser and Fieser, Reagents for .degree. runic Synthesis, John
Wiley & Sons, 1967; Protective Groups in Organic Chemistry,
McOmie J F W., Ed. Plenum Press, 1973; Chemical Reactions of
Polymers, Fettes E M., Ed. Intersience Publishers, New-York; Koenig
J L, Spectroscopy of Polymers, 2.sup.nd Ed. Elsevier, 1999). The
preferred mucoadhesive backbone is a polymer or a
polysaccharide.
[0090] The useful method of site-directed conjugation of antibody
molecules takes advantage of the carbohydrate chains attached to
the C.sub.H.sup.2 domain within the Fc region. Mild oxidation of
the polysaccharide sugar residues with sodium periodate will
generate aldehyde groups. A cross-linking or modification reagent
containing a hydrazide functional group then can be used to target
these formyl groups specifically for coupling to another molecule
(alginate backbone). Directed conjugation through antibody
carbohydrate chains thus avoids the antigen binding regions while
allowing for use of intact antibody molecules. This method often
results in the highest retention of antigen binding activity within
the ensuing conjugate.
[0091] As used herein, the term "antibody" is intended to include
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site that specifically binds an antigen (or a toxin), such
as Fab and F(ab').sub.2 fragments. As used herein, the term
includes polyclonal and monoclonal antibodies, and variants such as
single-chain (recombinant) antibodies, "humanized" chimeric
antibodies, and immunologically active fragments of antibodies. For
the purposes of this invention, a "chimeric" monoclonal antibody is
a murine monoclonal antibody comprising constant region fragments
(Fc) from a different animal. For the purposes of this invention, a
"humanized" monoclonal antibody is a murine monoclonal antibody in
which human protein sequences have been substituted for all the
murine protein sequences except for the murine complementarity
determining regions (CDR) of both the light and heavy chains.
Standard techniques for the generation and isolation of antibodies
are well-known and commonly employed by those of skill in the art.
A number of standard techniques are described in Kohler Milstein,
Nature 256:495-97 (1975); Kozbor et al., Immunol Today 4:72 (1983);
Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96
(Alan R. Liss, Inc., 1985); Kenneth, in Monoclonal Antibodies: A
New Dimension In Biological Analyses (Plenum Publishing Corp., New
York, N.Y. 1980); Lemer, Yale J. Biol. Med., 54:387-402 (1981);
Gefter et al., Somatic Cell Genet., 3:231-36 (1977); and Galfre et
al., Nature 266:55052 (1977).
[0092] As stated above, each mucoadhesive polymer can carry one or
more biologically active ligands linked to the polymer by a prodrug
linker or any other linker known to the person skilled in the art.
The polymers may have further substituents and may be further
functionalized. Non-limiting examples of such functional groups
comprise carboxylic acid and activated derivatives, amino,
maleimide, thiol, sulfonic acid and derivatives, carbonate and
derivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone,
hydrazine, isocyanate, isothiocyanate, phosphoric acid and
derivatives, phosphonic acid and derivatives, haloacetyl, alkyl
halides, acryloyl, arylating agents like aryl fluorides,
hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone,
vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide,
oxirane, and aziridine.
[0093] The averaged molecular weight of an antibody is on the order
of 150,000 Da while the averaged molecular weight of a monomer unit
of a polymer such as sodium alginate is 198 Da. Such a large
difference in molecular weights and size, along with a large
variety in structural conformations associated with the
macromolecule, e.g. the antibody, may result in steric hindrances
which will allow relatively low degree of molar modification.
Assuming the polymers to be mono-dispersed with an average
molecular weight of Mw=150,000 Da, and without wishing to be bound
by theory, it is believed that an efficient mucoadhesive ligand
would be one having between 1 and 3 antibody molecules per each
single polymer chain molecule, or in other words, a single
macromolecule for every 750 monomer unites of the polymer (150,000
Da/198 Da=750 monomer units). Assuming a ration of 3 macromolecules
per polymer, the percent coverage may be at least 0.4%. Using high
molecular weight polymer, being of, for example, 1,000.000 Da in
size allows a greater macromolecule converge in the order of
between 2 and 10%.
[0094] In case of low molecular weight ligands, comparable with
alginate monomer molecular weight the percent coverage is about 50%
of total functional group coupled.
[0095] Preferred functional groups for the polymer include but are
not limited to thiol, maleimide, amino, carboxylic acid and
derivatives, carbonate and derivatives, carbamate and derivatives,
aldehyde, and haloacetyl. Especially preferred functional groups
include thiol, maleimide, amino, carboxylic acid and derivatives,
carbamate and derivatives, and carbonate and derivatives
thereof.
[0096] The viscoelasticity of mucoadhesive backbone has a great
effect on its adhesiveness and cohesiveness. By adsorbing, swelling
and capillary action, the mucoadhesive backbone takes up water from
the mucus membrane, which leads to a strong adhesion. The
evaluation of a certain substance as potentially capable of strong
adhesiveness and long-mucus retention may be determined in
accordance with numerous tests known to a person skilled in the
art. The simplest test for adhesiveness is the tensiometry
experiment which measures the force required to detach two surfaces
(Biochemical Society Transactions, vol. 31, part 5, 1036-1040,
2003). The mucoadhesive property of the mucoadhesive ligands may
also be evaluated by an in vitro adhesion testing method known as
the wash-off method. The mucoadhesiveness of these ligand molecules
may be compared with that of a non-mucoadhesive material, such as
ethylene vinyl acetate.
[0097] The term "effective amount" as used herein refers generally
to the amount which is effective to remove from the mucosal
membrane a maximum amount of absorbed or adsorbed toxin which may
be sufficient to prevent adsorption or absorption of the a toxin
making contact with the membrane, ex vivo or in vivo, or to prevent
or delay the onset of a disease or disorder associated with said
toxin. The term "preventing" or any lingual variation thereof,
refers to precluding a disease or disorder which is associated with
an exposure to said toxin from occurring, or reducing symptoms
associated with an existing disease or disorder, or inducing a
pharmacological change or hygienic result relevant to treating the
disease or disorder, or minimizing complications and side effects
of a disease or disorder or the occurrence of a secondary disease
or disorder which may result therefrom, or arresting or delaying
the onset of clinical changes associated with such a disease or
disorder. The appropriate dosage for the pharmaceutical agents will
often be approximately comparable to that of the mucoadhesive
ligand alone; dosages may vary considering many factors including
age, weight, and condition of the subject needing such composition,
as well as the pharmacokinetics of the specific agent. The
composition of the invention may contain one or more mucoadhesive
ligand; however, their proportion in the composition will typically
be sufficient to procure the required therapeutic or hygienic
action.
[0098] In another aspect of the present invention there are
provided pharmaceutical compositions comprising any one of the
mucoadhesive ligands utilized with the method of present invention.
Also contemplated by the present invention are various uses for
such mucoadhesive ligands, i.e. as therapeutics and for
hygiene.
[0099] The compositions of the present invention may further
comprise one or more of a variety of agents such as for example pH
adjusters, carriers, excipients, diluents, antibiotics,
antioxidants, vehicles such as starch, microcrystalline cellulose,
lactose, sorbitol, or mannitol; lubricants such as magnesium
stearate, glycerol behenate, talc, hydrogenated ricin oil or waxes;
flow agents such as colloidal silica; aromas, flavoring agents,
sugaring or sweetening agents and in general any substances capable
of improving taste, odor or appearance of the composition, and one
or more pharmaceutical agents such as glucocorticoids,
dexamethasone, dexamethasone salts, isothiozolones, anticoagulants,
heparin, hirudin, peptides such as oligopeptides and polpeptides,
oligopeptides, antimitotic agents, angiopeptin, polynucleotides,
and oligonucleotides, sulfyhdryls, hydroxamic acids, oral
compositions including bioadhesive syrups and gels, mouth wash,
cough syrups and oral gels for mouth sores.
[0100] These agents may be hydrophobic, hydrophilic, or amphiphilic
in nature and can belong to a specific therapeutic class or within
an area covering cardiovascular, bronchodilation, enzyme
supplements, estrogen and androgen supplements, growth-stimulating
supplements, anti-parkinsonism memory maintenance, memory retention
or enhancement, anti-anxiety, antidepressant, birth-control,
antibiotic, antiviral, antiprotozoal, vitamin, antidiabetic,
gastro-intestinal, anticonvulsant, immunomodulation, nutritional
supplements and appetite modulating therapy.
[0101] These agents may be part of the composition itself or
alternatively may be synthetically attached to the mucoadhesive
ligand. For example, in one case the polymer mucoadhesive ligand
may have an appetite-modulating agent attached thereto. When
applied to the mucosal membrane, the polymeric backbone releases
said agent without affecting its ability to attach to toxin
molecules present in the membrane.
[0102] Suitable pH adjusting substances include any such substance
that is safe for mammalian use. More preferably, the pH adjusting
substances include any weak acid or weak base, e.g., sodium
carbonate, potassium carbonate, disodium hydrogen phosphate, sodium
dihydrogen phosphate, and the equivalent potassium salts.
[0103] Examples of peptides are, without being limited thereto,
cytokines, proteins, enzymes, hormones, monoclonal antibodies,
human growth hormones, clotting factors, colony stimulating
factors, erythropoietins, tissue plasminogen activators,
recombinant soluble receptors, anti-bacterial agents,
anti-neoplastic agents, anti-fungal agents, immunomodulators,
antiparasitic agents, CNS agents and vaccines.
[0104] Examples of polypeptides, without being limited thereto, are
antibodies, immunomodulators or cytokines, e.g. interferons or
interleukins, peptide hormones, e.g. colony stimulating factors and
tumor necrosis factors, hormone receptors, neuropeptides,
lipoproteins, erythropoietins, growth hormones, thyroid hormones,
toxins such as diphtheria toxin, proteoglycans such as hyaluronic
acid, and glycoproteins such as gonadotropin hormone.
[0105] The pharmaceutical composition comprising of the
mucoadhesive ligand may be administered also in conjunction with
enzyme inhibiting agents such as reverse transcriptase inhibitors,
protease inhibitors, angiotensin converting enzymes,
5-alpha-reductase, and the like. Typical agents include peptide and
nonpeptide agents including finasteride, lisinopril, saquinavir,
quinapril, ramipril, indinavir, ritonavir, nelfinavir, zalcitabine,
zidovudine, allophenylnorstatine, kynostatin, delaviridine,
bis-tetrahydrofuran ligands, and didanosine.
[0106] The pharmaceutical composition of the present invention may
be formulated in the form of suppositories, tablets, films,
patches, and gels for oral, buccal, nasal, ocular, and topical
routes; as well as spray formulations, drops, dry powder,
suspensions and the like. The composition may additionally be
formulated as microparticles.
[0107] The method of the present invention comprises administering
to a subject an effective amount of a pharmaceutical composition
comprising a mucoadhesive compound. Within the scope of the present
invention, said mucoadhesive compound may not necessitate the
presence of ligand or chelator groups in order to capture and bind
to toxins. The ability of such mucoadhesive compound may rise from
structural characteristics such as folding of the compound in the
mucus layer thereby capturing the toxin irreversibly, the presence
of pockets or cavities within the folded polymer, which may trap
the toxin irreversibly, low-attraction forces etc.
[0108] Without wishing to be bound by theory, the removal of toxins
from a mucosal membrane may take place as follows: upon application
of the composition comprising the mucoadhesive compound, the buccal
liquid e.g. saliva or nasal liquid or any other fluid which covers
the treated mucosal membrane, penetrates into the composition and
hydrates the mucoadhesive polymer, leading to the formation of a
matrix. Such buccal liquid, which may contain the toxins, come into
contact with the matrix of the mucoadhesive ligand, and
progressively become bonded or attached thereto. The mucoadhesive
agent may than be expelled from the mucosa membrane due to one or
more of the following possible occurrences: (1) due to loading with
the toxin, the ligand's mucoadhesivity reduces and it becomes
detached from the layer or clue to release of reversibly chelated
ligand D (as described hereinbefore) which creates changes in the
environment of mucoadhesive polymer reducing polymer's mucoadhesive
properties; (2) it is no longer retentive; (3) it is expelled with
the secretion of the mucus from the mucus glands; (4) it may be
physically withdrawn from the mucus layer, e.g. by a tooth brush;
(5) it may be absorbed into the blood system and expelled through
the urinary or GI tract; (6) it may be vigorously washed off; (7)
it may be biodegradable; or (7) in case of the nose and respiratory
system it may be removed therefrom by sneezing and/or coughing.
Example 1
Preparation of Oxidized Antibody (FIG. 3, Step Alfa))
[0109] The antibody to be periodate-oxidized is dissolved at a
concentration of 10 mg/ml in 0.01 M sodium phosphate, 0.15 M NaCl,
pH 7.2. Sodium periodate is dissolved in water to a final
concentration of 0.1 M and protected from light. Immediately
thereafter 100 .mu.l of the sodium periodate solution are added to
each milliliter of the antibody solution and allowed to react in
the dark for 30 ruin at room temperature. The oxidized antibody is
purified by gel filtration using a column of Sephadex G-25. The
chromatography buffer is 0.1 M sodium phosphate, 0.15 M NaCl, pH
7.2. To obtain efficient separation between the oxidized antibody
and excess periodate, the sample size applied to the column should
be at a ratio of no more than 5% sample volume to the total, column
volume. Collect 0.5-ml fractions and monitor for protein at 280 nm.
Pool the fractions containing protein. Adjust the antibody
concentration to 10 mg/ml for the conjugation step. The oxidized
antibody should be used immediately.
Example 2
Coupling of Oxidized Antibody with Adipic Acid Dihydrazide (FIG. 3,
Step A1(b))
[0110] Dissolve a macromolecule (such as a protein/antibody)
containing aldehyde functional groups (obtained in previous step)
in a buffered solution at a pH of about 7-8.5 and at a
concentration of about 1-10 mg/ml, Phosphate, carbonate, borate, or
similar buffers adjusted to this pH range work well. Avoid
amine-containing buffers (i.e., glycine or Tris) or other
components containing strong nucleophiles, since these may react
with the aldehydes. Higher pH environments enhance the formation of
hydrazone bonds and generally increase the yield of complex. Add a
quantity of adipic acid dihydrazide (Aldrich) to the protein
(antibody) solution to obtain at least a 10-fold molar excess over
the amount of aldehyde functional group present. If the
concentration of aldehydes is unknown, the addition of 32 mg adipic
acid dihydrazide per milliliter of the protein solution to be
modified should be used. React for 2 h at room temperature.
Although hydrazone formation does not require the addition of a
reductant to create a linkage, including sodium cyanoborohydride
(NaCNBH.sub.3) in the reaction mixture considerably increases the
yield and stability of bonds formed. If the presence of a reducing
agent will not cause harm to the macromolecule being modified, the
addition of 10 .mu.l of 5 M sodium cyanoborohydride (Sigma) per
milliliter of reaction solution may be done. Purify the modified
protein (antibody) by dialysis or gel filtration.
Hydrazide-activated proteins are stable to long-term storage at
4.degree. C. in the presence of a preservative (0.05% sodium azide)
or in a frozen or lyophilized state.
Example 3
Coupling of Hydrazide Activated Antibody to Alginate (FIG. 3, step
A2)
[0111] Dissolve 50 mg of hydrazide activated antibody in 1% (w/v)
solution of alginate (20 ml solution, 1.0 mmol alginate monomer) in
0.1 M MES buffer, pH 6.0. Then, add 0.0216 g (0.1 mmol) of NHSS and
0.0384 g (0.2 mmol) of EDAC (ratios of reagents were calculated for
a theoretical 20% molar modification of the number of carboxylic
groups of alginate). React for 3 h at room temperature and purify
the final product by reversible precipitation of alginate by
decreasing the pH up to 3. The precipitate was collected by
centrifugation, washed with water and then resolubilized in 0.1 M
MES buffer. Alternatively, removal of non bound-antibody was
accomplished by precipitating the alginate with 1% calcium chloride
solution. The precipitate was collected by centrifugation, washed
with water and resolubilized by addition of 0.1M Tris buffer pH 7.5
and a sufficient amount of 0.4M sodium citrate pH 6.3 to permit
complete solubility.
[0112] Alternatively, alginate can be first modified with adipic
dihydrazide using EDAC/NHSS activation of alginate and 10 molar
excess of dihydrazide to prevent alginate self-crosslinking. The
hydrazide activated alginate could be purified by dialysis. Then,
sodium periodate oxidized antibody (described above) could be
coupled to alginate by forming the hydrazone bonds. The final
product will be purified by reversible precipitation as described
above. .sup.13C NMR and FTIR spectroscopy could be used to confirm
the hydrazide activated alginate. The final antibody modified
alginate could by analyzed using absorption method at 280 nm for
protein content determination, ELISA or colorimetric protein
determination method (Micro BCA.TM. Protein Assay Kit, cat. number
23235, Pierce) as well.
[0113] In the second approach, alginate could be coupled directly
with antibody via carbodiimide chemistry as shown in FIG. 3, step
B). In this case antibody is coupled through its amino groups to
alginate backbone. The purification of antibody-modified polymer
could be performed as described above.
Example 4
Preparation of Biotin-Conjugated Alginate in Aqueous Media
[0114] The biotin-alginate conjugate was prepared as shown in FIG.
2, step A. An amount of 0.052 g (0.2 mmol) of biotin hydrazide was
added to a 1% (w/v) solution of alginate (20 ml solution, 1.0 mmol
alginate monomer) in 0.1 M MES buffer, pH 6.0. The reaction mixture
was stirred at room temperature for 50 min to facilitate a
homogeneous dispersion of the biotinylating reagent in the reaction
solution. Then, 0.0216 g (0.1 mmol) of NHSS and 0.0384 g (0.2 mmol)
of EDAC were added (ratios of reagents were calculated for a
theoretical 20% molar modification of the number of carboxylic
groups of alginate). After 3 h at room temperature, the resulting
polymer was dialyzed against doubly deionized water using a 10,000
MWCO membrane (66410, Rockford, USA). The water was changed twice a
day for three days, after which time the modified alginate was
lyophilized.
[0115] Spectroscopic Analysis
[0116] For FTIR spectroscopy, polymer samples were prepared as thin
films as follows: 4 mg/ml of the modified alginate was dissolved in
doubly deionized water. The resulting solution was poured into a
polystyrene Petri dish and dried in an oven at 50.degree. C. for 24
h to produce a thin transparent polymer film.
[0117] Quantitative Assay of the Extent of Biotinylation of the
Alginate
[0118] A fluorescence-based method was used to determine the
available biotin content in the modified alginate. In the presence
of avidin, the fluorescence of 2,6-ANS is blue shifted (from 463 to
422 nm) with a large increase in quantum yield. Biotin binding
causes complete displacement of the bound fluorophore, with a
concomitant quenching of the fluorescence. The fluorescence-based
assay was adapted to a 96-well microtitration plate format Volumes
of 1 .mu.L of a 2,6-ANS (6 mg/mL in DMSO and 66 .mu.L of avidin (2
mg/mL) were added to different volumes of biotin (0.1 ml) from 0 to
120 .mu.L (in increments of 10 .mu.L) in the wells of a
microtitration plate. Each well was brought up to a total volume of
200 .mu.L with PBS, pH 6.0. Biotin-alginate samples (1 mg/mL) in
volumes from 2 to 20 .mu.L (in increments of 2 .mu.L) were then
assayed to determine the amounts of biotin on the alginate
available for complexing with avidin. The fluorescence was
monitored at 320 nm (excitation) and 405 nm (emission) by a
POLARStar Galaxy fluorimeter (BMG-labtechnologies GmbH, Germany).
All solutions, except 2,6-ANS, were prepared in PBS at pH 6.0.
Example 5
Activation of Alginic Acid with N-Hydroxysuccinimide and DCC in
Organic Media
[0119] A 1.20 g (10.4 mmol) N-hydroxysuccinimide (NHS, Mw=115
g/mol) and 50.0 mmol (8.80 g) Alginic Acid (176 g/mol of monomer
unit) dissolved in 100-150 ml of dry N,N-dimethylformamide (DMF) in
a dry Argon atmosphere. After addition of 2.06 g (10.0 mmol)
N,N'-dicyclohexylcarbodiimide (DCC, Mw=206.33 g/mol) the solution
stirred overnight at room temperature. The precipitated solid
filtered of and the filter rinsed with DMF or Acetonitrile-Acetone
(1:1), and the filtrate diluted with a mixture of petroleum ether
(Merck, 40-60.degree. C.) and Isopropanol (6:1). After 2 h at room
temperature a crude product is expected to be recovered by
filtration, redissolved DMF and recrystallized from Petroleum
ether:Isopropanol (6:1) to yield white solid product after drying
in vacuum. Analysis must be performed. This protocol for activation
of alginate is useful if the next coupling step will be carried out
in the organic media. This protocol can farther be used for
obtaining aldehyde activated alginate for ligand protein coupling
(using in the final step 10% Dimethylformamide (DMF) or
dimethylsulfuoxide (DMSO) in aqueous media for preservation of
ligand-protein activity. Additional use of this activation of
alginate could be used for fluorescence labeling by BODIPY
(Molecular Probes).
Example 6
Preparation of Benzaldehyde Conjugated Alginate (FIG. 4, Steps
B1-B3)
[0120] In order to prepare a benzaldehyde conjugated alginate, a
few appropriate precursors are first synthesized.
4-(2-bromoethoxy)benzaldehyde is prepared by reacting
p-hydroxibezaldehyde with 1,2-dibromoethane, in dry acetone. The
product is then reacted with ethyleneglycol in toluene, with para
toluene sulfonic acid as a catalyst, in dry conditions, in order to
afford protection to the labile aldehyde groups. The protected
2-(4-(2-bromoethoxy)phenyl)-1,3-dioxolane is then reacted with
potassium phthalimide, to afford
2-(4-(1,3-dioxolan-2-yl)phenoxy)ethanamine which is next coupled to
the alginate using carbodiimide chemistry (using EDAC and NHSS as
activators for alginate), as described previously. Then, the
protecting group on the 1,3-dioxolane is removed by ppTs, in
ethanol, and the free aldehyde is obtained, Any ligand protein may
now be coupled by creating a Schiff base bond between the activated
alginate and the amino group of the protein.
Example 7
Methods for the Quantification of the Extent of Alginate
Modification with the "Ligand" Antibody or Determination of Binding
Capacity of Modified Polymer
[0121] 1. Direct Method
[0122] To modify the alginate with fluorescently labeled antibody
and to measure the fluorescence of modified alginate. The extent of
alginate modification could be calculated from the calibration
curve obtained from measurement of the fluorescence of standard
solutions of fluorescently labeled antibody (FIG. 5).
[0123] 2. Indirect Methods
[0124] To conjugate the antibody modified alginate with its
fluorescently labeled target virus and to measure the fluorescence
of the bound virus. To perform this assay the alginate should be
immobilized on the solid surface to allow blocking and washing
steps before and after virus binding. The immobilization of
alginate (polyanion) on the solid support could be achieved using
polylysine (polycation) coated 96-well plates.
[0125] The mono/bis NHS-ester of Cy3 fluorescent dye (Amersham
Biosciences Cat. No PA 23001/23000 can be used to fluorescently
label the "target" virus (FIG. 6).
[0126] Alternative non-direct assay could be based on the sandwich
ELISA principle. In the first step, to couple with antibody
modified alginate its target virus. Then to introduce fluorescently
labeled primary to target virus antibody. In that case also a
calibration curve should be used to derive the extent of
modification of alginate. Furthermore, this non-direct assay could
allow calculating the real capacity of modified alginate to bind
the "target" virus. In, that case, also a surface immobilization of
alginate will be necessary. An alternative immobilization of
alginate on the solid surface could be achieved using biotin
modified alginate and avidin coated surface as summarized in the
section describing the kinetic studies (FIG. 7).
Example 8
Binding Studies to Toxicants
[0127] For the binding and retention kinetics studies of various
target entities the phenomenon of surface plasmon resonance (SPR)
may be used to monitor the biomolecular binding events in real time
without the use of labels.
[0128] In general, the refractive index change for a given change
of mass concentration at the surface layer, is practically the same
for all proteins and peptides, and is similar for glycoproteins,
lipids and nucleic acids. Biomolecular binding events cause further
changes in the refractive index and the SPR signal.
[0129] The kinetics of an interaction, i.e. the rates of complex
formation (K.sub.a) and dissociation (K.sub.d), can also be
determined as known to a person skilled in the art. The affinity of
an interaction is determined from the level of binding at
equilibrium (seen as a constant signal) as a function of sample
concentration. Affinity can also be determined from kinetic
measurements. For a simple 1:1 interaction, the equilibrium
constant K.sub.D is the ratio of the kinetic rate constants,
K.sub.d/K.sub.a. The extent to which different molecules interact
with a single partner immobilized on a sensor surface reveals the
specificity of an interaction. Simple yes/no answers are able to
allow search for binding partners and performing of test for
cross-reactivity.
[0130] For the kinetic studies, a dually modified alginate may also
be used. First modification with biotin to the extent of 5-10% is
necessary to link the alginate with the avidin coated chip surface.
Second modification with appropriate ligand will be used for
kinetic/affinity studies. The target molecule will flow over the
chip surface interacting with the ligand immobilized on the chip
(FIG. 8A). Alternatively, ligand modified alginate could be
immobilized on the amino coated chip surface via carbodiimide
chemistry using EDAC/NHSS activation for alginate.
[0131] Alternative binding/retention kinetic studies could be
performed using fluorescently labeled target molecules. In that
case, again dually modified alginate could be used. Avidin-biotin
interaction allows for alginate immobilization on the surface of
the glass/gold coated slides by means of silane/thiol chemistry. To
obtain the kinetics data in that case the depletion (FIG. 8B) or
the enrichment (FIG. 9B) of fluorescence of the "target" molecule
in the solution flowed over the slide could be measured. For the
retention studies, all binding sites of the alginate should be
blocked or saturated with "target" molecule. Then, by passing fresh
buffer to elute the "target" molecule the enrichment in
fluorescence could be measured and retention kinetic could be
determined.
Example 9
Gastric Juice Retention (Biodegradation) Studies
[0132] Preparation of Degradation Media
[0133] Simulated gastric fluid (SGF) and simulated intestinal fluid
(SIF) may be prepared in accordance with known procedures.
Typically, the SGF is prepared by dissolving 2 g sodium chloride
and 3.2 g pepsin (Sigma) in 7 ml, hydrochloric acid and volume
adjusted to 1 L by water to obtain a pH about 1.2. The SIP is
prepared by dissolving 6.8 g of monobasic potassium phosphate in
250 mL of water. The solution is mixed and 190 mL of 0.2 N sodium
hydroxide and 400 mL of water and 10 g of pancreatin (Sigma) are
added. The pH then adjusted with 0.2 N sodium hydroxide to
7.5.+-.0.1 and the volume completed to 1 L by water. Antibiotics
such as Penicillin-streptomycin and antimycotic such as Fungizone
may be added to each solution to avoid bacterial contamination.
[0134] Biodegradation Studies
[0135] To study the biodegradation of ligand (antibody) modified
alginate, a hydrogel microspheres may be prepared using the gas
shear method, which employs a concentric air stream to shear the
drop from the needle tip controlled essentially by the gas
flow.
[0136] A 100 mg of dried microspheres are suspended in 5 mL of each
medium (SGF and SIF) in 15 mL plastic tubes. Sample tubes are
incubated at 37.degree. C. under continuous shaking at 240 rpm.
After a predetermined incubation time, the solution is filtered
trough 0.45 mm Millipore nylon filter for HPLC and MALLS-SEC
(multi-angle laser light scattering and size exclusion
chromatography) analyses. Using HPLC analysis, the protein
(antibody) degradation could be assessed. Using MALLS analysis, the
carbohydrate degradation could be studied by measuring molecular
weight distribution of the fragments of digested polymer.
[0137] Scanning Electron Microscopy (SEM)
[0138] To determine the microspheres morphological and
ultrastructural changes after incubation, scanning electron
microscopy could be used. For SEM observations, incubated
microspheres after degradation should be washed three times with DD
water and stabilized with 3% glutaraldehyde solution in a 0.1 M
phosphate buffer at 4.degree. C. for 60 min. The microspheres then
should be rinsed several times with the 0.1M phosphate buffer
solution and post-fixed in 1% Osmium. Tetroxide (OsO.sub.4) in a
0.1 M phosphate buffer at pH 7.2 for 24 at 4.degree. C. After
rinsing with water, the microspheres are dehydrated sequentially
with increasing ethanol solutions concentrations (30, 50, 70, 90,
95 and 100 vol %). The ethanol-impregnated gel samples were totally
dried by the critical point drying method (CDP). The dried samples
fixed on aluminum sample holders using a silver-based adhesive and
then coated by a conductive layer (15 nm) of Au/Pd (sputtering
method).
Example 10
In Vitro Retention Strength Studies of Biopolymer (Mucoadhesion
Studies)
[0139] Rheology as Means of Evaluating Polymer-Mucin
Interactions
[0140] This approach is, to some extent, based on the
interdiffusion-interpenetration theory of the mucoadhesion process
and, as such, aims to simulate the interpenetration layer between
the gel and the mucus layer. When a putative mucoadhesive polymer
is mixed with a mucin solution, there is a synergistic increase in
viscosity. It is known that the viscosity of a polymer-mucin
mixture should be considered to be the result of the contributions
from the separate components, the polymer and mucin, and from a
viscosity component arising from mechanical interactions
(entanglements) and chemical interactions between the polymer and
the mucin. For mucoadhesive polymers it is believed that the
theological response of a polymer-mucin mixture should be larger
than the sum of the contributions from the gel and the mucin, a
phenomenon that is commonly described as "rheological
synergism".
[0141] The Synergism Parameters
[0142] The most commonly used synergism parameter, .DELTA.G', also
called the interaction term, is the elastic component that is
calculated from the equation
G'.sub.mix=G'.sub.p+G'.sub.m+.DELTA.G', wherein G'.sub.mix is the
elastic modulus of the polymer-mucin mixture and G'.sub.p and
G'.sub.m represent the elastic modulus of the polymer and mucin
respectively. This equation may be simplified because of the
negligibly small elastic modulus of the mucin solutions at certain
concentration range. Therefore, .DELTA.G' could be calculated from:
C'.sub.mix=G'.sub.p+.DELTA.G'.
[0143] Furthermore, the relative synergism parameter, which has
been put forward as alternative to the absolute synergism parameter
may be calculated from:
Relative .DELTA. G ' = .DELTA. G ' G p ' = G mix ' - G p ' G p ' .
##EQU00001##
[0144] A rheometric measurement normally consists of a strain
(deformation) or a stress analysis at a constant frequency
(normally 1 Hz) combined with a frequency analysis, e.g. between
0.1 and 100 Hz. The strain sweep gives information of the elastic
modulus (Y, the viscous modulus G'' and the phase angle. A large
value of G' in comparison of G'' indicates pronounced elastic (gel)
properties of the product being analyzed. For such a product the
phase angle is also small, e.g. 20.degree. (a phase angle of
0.degree. means a perfectly elastic material and a phase angle of
90.degree. means a perfectly viscous material). The frequency sweep
gives information about the gel strength where a large slope of the
G' curve indicates low strength and a small slope indicates high
strength.
[0145] A viscometric measurement normally consists of a shear rate
analysis. The shear rate sweep should preferably cover the range
applied in the intended equipment. For liquid samples a shear rate
range from around 1 to 1,000 s.sup.-1 covers the needs for a
low-viscous materials and a shear rate range from around 1 to 100
s.sup.-1 covers the needs for a high-viscous materials.
[0146] Tensile Strength Methods for Measuring the Mucoadhesion of
Gels
[0147] There are two ways by which the mucoadhesion of the
compounds may be evaluated. In the first approach, the measurement
configuration involved one piece of mucosa and a large volume of
agent to be tested.
[0148] In the other approach, a relatively small volume of the
agent is used and placed between two pieces of mucosa. In this
method, the measurement started by lowering the upper mucosa until
contact was made with the agent. After a certain contact time the
upper mucosa was slowly withdrawn upwards at a constant speed until
detachment occurred. During the entire measurement a force-distance
curve was recorded from which the tensile work (i.e., the area
under the curve during the withdrawal), the peak force and the
deformation to failure were determined. As the mucosa is separated
from the agent (preferably in a gellous form), failure will occur
in the weakest of the three regions of the mucoadhesive complex: in
the gel, in the mucus or in the interface layer between the gel and
the mucus where it is possible that interactions strengthen the
mucus layer. Consequently, the force-distance curve recorded in the
measurement gives a measure of the strength of the bonds in the
weakest region. To interpret the results and to determine the
region in which failure occurs (i.e., which bonds are reflected in
the acquired data), the cohesiveness of these components with the
results from the mucoadhesion measurement should help to identify
which region is the weakest. This procedure offers a good basis
from which to asses whether the measured tensile work reflects a
genuine interaction of the gel preparation with the mucus layer or
the cohesive failure of the gel.
[0149] Alternative In-Vitro Mucoadhesion Studies
[0150] Another method to measure of mucoadhesive properties of
modified alginate microspheres is by determination of the quantity
of microspheres sticking to a filter paper saturated with mucin and
after applying an air load. The air jet can be used to simulate
breathing in and out. It could be useful to evaluate the effect of
air-flow on nasal clearance of micro particulate formulations after
their administration.
Example 11
In Vivo Feasibility Experiment to Trap the Model
Pathogen-Adenovirus (AdV) Aerosolized into Mouse/Rat Oral Cavity
Pre-Coated with Adhesive Bio-Hybrid Polymer-Alginate
[0151] The purpose of alginate modification with anti-AdV antibody
is to provide the alginate the ability to bind free adenovirus
(AdV). The purpose of alginate modification with cystein or wheat
germ agglutinin lectin (WGA) is to provide the alginate with
improved mucoadhesive properties for better retention of the
polymer on mucoadhesive tissue, e.g. in the mouth cavity (buccal
cavity) and larynx or upper throat.
[0152] Alternatively, two mono-modified alginates can be used in
mixture: Alginate modified with AdV antibody or Alginate modified
with cystein/lectin (WGA). The mixture of alginates may be used to
obtain conceptually similar properties (affinity to AdV and
improved mucoadhesive ability) as could be achieved by dually
modified alginate described above.
[0153] Alternatively, mono-modified alginate with anti-AdV antibody
as initial test without the enhancing the mucoadhesive properties
may also be tested.
[0154] The mucoadhesive polymers of the invention may be
cross-linked in situ after administration to the target membrane.
In such an approach, the polymer is aerosolized (sprayed) in the
mouth cavity and the upper respiratory tract in an anesthetized
animal. This forms the initial film of non cross-linked polymer
solution in the buccal cavity and the respiratory tract.
Immediately thereafter, a solution of crosslinker is sprayed in the
mouth cavity and respiratory tract of animal. In this way, the
deposited polymer(s) is crosslinked on the interior oral cavity
surface creating biohybrid hydrogel coating. The additional number
of hydrogel layers could be created on the base of the first
hydrogel layer by the same repeatable manipulations: spraying of
additional polymer dose and a consequent spraying of the
crosslinker reagent to harden the gel. The purpose of few layers is
to create a homogeneous coating in the oral cavity of the animal.
The number of polymer layers may be determined experimentally.
[0155] The capturing of the AdV may be tested as follows: The
control and the experimental groups (pre-coated with
non-functionalized alginate and functionalized alginates
respectively) of mildly anesthetized animals are exposed to the AdV
spray (aerosol) using Nebuliser. Additional animal control groups
are: (1) animal without protective polymer coating exposed to the
same AdV dose, (2) non-treated animals without the gel pre-coating
and without the exposure to AdV and (3) pre-coated animals with
protective polymer but not exposed to AdV. Control (2) and (3) will
allow to determine the background fluorescence of the non treated
animal and effect of gel on animal respectively.
[0156] Both the time of exposure to AdV and the appropriate viral
titer are determined experimentally. 24-48 hours post exposure to
AdV, the animals are imaged non-invasively using an imaging
system.
[0157] In addition, to follow up alter the expression of reporter
genes encoded by AdV it is also possible to track the AdV
localization in the animal by using fluorescently labeled AdV,
using for example the mono/bis NHS-ester of Cy3 fluorescent dye.
Tracking of AdV in animal could be assessed using non invasive
imaging mentioned above or fluorescent microscopy.
Example 12
Mouse Lung Lavage: Determination of the Persistence of Hydrogel in
the Lungs
[0158] Mice are killed by CO.sub.2 suffocation by placing them in a
jar containing dry ice. The asphyxiated mouse is then placed on its
back and its ventral side is swabbed with ethanol. A midline
ventral incision is made and the fir stretched away from the
abdomen to the mandible, uncovering the muscle layer. The
sternohyoid and throat muscles are teased apart; the trachea is
exposed and left hi-situ. The thoracic cage is partially removed to
uncover the intact lungs. A thread is placed just under the trachea
and bath sides of it are used to slightly pull the trachea up. A
Venflon.RTM. I.V. Cannula is inserted into the trachea towards the
lungs within 5 mm of the larynx. The injection valve is slowly
pulled out leaving the Teflon catheter inserted. The cannula is
firmly secured to the trachea with single knot to avoid leakage and
then attached to a three-way stopcock, previously connected to two
syringes, one empty and the other containing BSA/PBS. Two to three
flushes are used to rinse the inflated lungs and the aspirated
bronchial secretions are drawn into the other syringe. The
collected bronchoalveolar lavages are pooled, dispensed into
aliquots, and then stored at -20.degree. C. until assayed. If
needed, serum can also be obtained by cutting a nearby artery and
the heart. Just before use, the washings are centrifuged to remove
cellular debris and pooled samples containing erythrocytes are
discarded.
Example 13
Ligated Ileal Loop: Protective Capacity of the Bioaffinity Hydrogel
Linked with Anti-Cholera Toxin Antibodies
[0159] Anesthetized postweaned rabbits (4-8 months old, 1-2 kg) are
put on a liquid diet 48 h prior to the challenge assay. A 100 cm
piece of small bowel beginning just above the appendix is exposed
and kept moist while experimental (6 cm) and spacer (2 cm) loops
are tied. Fully enteropathogenic Vibrio cholarae strain 395 is
slowly injected into the large loops in ten-fold serial dilutions
with PBS as a control. A second tie is made to isolate the site of
injection. Before closing the abdomen in one layer with a running
stitch, 5% glucose is given i.p. to reduce postoperative
dehydration and the skin is then clipped shut. Food is not made
available after the operation but water is supplied ad Libitum. The
rabbit is killed after 18-20 h. Pictures are taken of the dissected
small intestine containing the loops, and labeled according to
contents and anatomy. Leakage between positive and negative loops
is checked by injecting Pontamine sky blue 6 BX. Negative loop
reactions are indistinguishable from normal collapsed bowel, while
the positive loops are elongated and distended with fluid
containing flecks of mucus, vibrios, enterocytes and other
biological material. The fluid accumulation is measured by
liberating the turbid contents from the tightly distended sacs.
Example 14
IVIS--Nasal to GI Tracing of a Hydrogel Preparation in Mice Using
IVIS System
[0160] 15 Balb/C mice (8-10 weeks old) will be put on a chlorophyll
free diet 72 h prior to the challenge assay. The mice will be
anesthetized and fur removed (by shaving or depilation) and the
mice will be divided into 5 groups. Group C (calibration) will be
anesthetized. Then a calibration test will be conducted for the
purpose of determining the right concentration of Cy7-avidin to be
detected by the IVIS system. Each of the other groups will be
anesthetized and will be provided with different formulations
through nasal instillation and then will be examined using the IVIS
system: Group I will be provided with the buffer solution
(solvent), Group II will be provided with Cy7-avidin, Group M will
be provided with conjugated alginate biotin, Group IV will be
provided with conjugated alginate-biotin bound to Cy7-avidin. The
concentrations will be determined by the calibration assay (group
C). The mice will be detected by the IVIS imaging system, three
mice at a time. All animals will be euthanized by respiratory
exposure to excess CO.sub.2.
Example 15
Toxin Challenge Model: Demonstration that Bioaffinity Hydrogel is
Protective Against a Model Toxin
[0161] 40 Balb/C mice (8-10 weeks old) will be divided into fbur
groups, with two sub-groups in each group. The animals will be
provided through nasal instillation with staphylococcus enterotoxin
33, dissolved in sterile pyrogen-free phosphate buffered saline.
Group I will be provided with only buffer solution (control), group
II will be provided with 0.25 .mu.g toxin per every gram of the
mouse weight, group III--0.75 .mu.g/gr, group IV--1 .mu.g/gr. All
the instillations will be provided by the equation of 1.5 .mu.l/gr.
Every sub group will be euthanized 24 h after the instillation by
respiratory exposure to excess CO.sub.2. Airway lavage will be
performed in the euthanized animals, and the number of inflammatory
cells will be counted. The other sub-group will be supervised over
four days, to test the influence of the toxin. The animals will be
weighed every day during the supervision. Clinical signs like
hypothermia, inactivation and loss of more than 20% in body weight,
will be watched for 2-3 times a day. The schedule of the sacrifice
may be brought forward if the animal shows clinical signs of severe
ill health, before the determined euthanasia of 96 h after the
toxin exposure. All animals will be euthanized by respiratory
exposure to excess CO.sub.2. Airway lavage will be performed in
animals euthanized 96 h after toxin administration and the number
of inflammatory cells will be counted.
Example 16
In Vivo Studies: Challenge Studies with Virus
[0162] 40 Balb/C mice (8-10 weeks old) will be divided to four
groups, with four sub-groups in each group. The animals will be
provided through nasal instillation with influenza virus. Group 1
will be provided with only buffer solution (control), group 2 will
be provided with 0.25 .mu.g toxin per every gram of the mouse
weight, group 3 with 0.75 .mu.l/gr, group 4 with 1 .mu.g/gr. All
the instillations will be provided by the equation of 13 .mu.l/gr.
Every sub group will be euthanized 24 hours after the instillation
by respiratory exposure to excess CO.sub.2. Airway lavage is
performed in animals euthanized, and the number of inflammatory
cells is counted.
[0163] The other sub-group will be supervised over four days, to
test the influence of the toxin. The animals will be weighed every
day during the supervision. Clinical signs like hypothermia,
inactivation and loss of more than 20% in body weight, will be
observed 2-3 times a day. The schedule of the sacrifice might be
brought forward if the animal shows clinical signs of severe ill
health, before the determined euthanasia of 96 hours after the
toxin exposure.
[0164] At the time of the scheduled sacrifice, all animals will be
euthanized by respiratory exposure to excess CO.sub.2. Airway
lavage is performed in animals euthanized 96 hours after toxin
administration and the number of inflammatory cells is counted.
Similar and appropriate protocols will be followed for the
instillation of rats with Rift Valley fever and of guinea pigs with
Marburg virus.
Example 17
Immobilization of Pollen and Viruses in a Hydrogel Composed of
Alginate Bio-Conjugated to Macromolecules with Non-Specific
Affinity and Adhesion Properties to Pollen and Viruses
[0165] Flowering plants possess in the female organs of the flower
specialized extracellular matrices that support pollen tube growth
and sperm cell transfer along the transmitting tract of the
gynoecium. Transport of the pollen tube cell and the sperm cells
involves a cell adhesion and migration event, in species such as
lily that possesses a transmitting tract epidermis in the stigma,
style, and ovary. A bioassay for adhesion was used to isolate from
lily stigma/stylar exudate the components that are responsible for
in vivo pollen tube adhesion. At least two stylar components are
necessary for adhesion: the first being a large molecule and the
second being a small (9 kD) protein (Stigma/stylar cysteine rich
adhesion, SCA). In combination, the two molecules induced adhesion
of pollen tubes to an artificial stylar matrix in vitro. The 9-kD
protein was purified, and its corresponding cDNA was cloned. This
molecule shows some similarity with plant lipid transfer proteins.
Immunolocalization data support its role in facilitating adhesion
of pollen tubes to the stylar transmitting tract epidermis.
1) Isolation of Lily Stylar Exudates:
[0166] Easter lily (Lilium longiflorum Thumb cvs. Snow Queen and
Nellie white) flowers should be collected 1-2 days after anthesis.
The pollen grains should be used immediately or dried at room
temperature overnight then stored at -80 degrees Celsius for
further use. The stigma exudate should be collected from the cut
stigma by incubation with an extraction buffer (see below). The
stylar exudates should be eluted by two different methods. Lily
styles should be collected and the stigma and ovary should be
removed. In one method, after removing the stigma and ovary the
stylar exudate should be eluted from the hollow style by applying
extraction buffer (84 mM citric acid, 2 mM Na2S2O4, pH 3) to the
top (the stigma position) of the style and collecting exudate from
the bottom (the ovary position). These exudates should be dried by
speed vacuum or lyophilization and stored at -20 degrees Celsius
for further characterization. A second method can be used: The
stigma and ovary of the lily gynoecium should be removed and the
style bisected longitudinally to expose the transmitting tract
tissue.
[0167] Stylae Segments of Lily gynoaeum 2-3 days after anthesis
should be incubated with the same extraction buffer for 2 hours at
4 degrees Celsius with gentle shaking the element should be
centrifuged at 6000 g for 10 min. to remove cell and/or tissue
debris. The eluted stylar exudates should be dialyzed against water
with Spectra/Por dialysis membrane tubing (MWCO 12,000-14,000,
Spectrum, Houston, TEX) for 2 days with at least four changes of
water. After dialysis the stylar exudates should be lyophilized and
stored at -20 Degrees Celsius separately for further
characterization and fractionation.
2) SDS-Polyacrylamide Gel Electrophoresis:
[0168] The crude lily stylar samples collected by two different
methods should be mixed with an equal amount of 2.times.SDS sample
loading buffer [100 mM Tris (pH 6.8), 100 mM beta-mercaptoethanol,
4% (w/v) SDS, 0.2% (w/v) bromophenol blue, and 20% (v/v) glycerol],
boiled for 5 min and fractionated by 12% SDS-poly-acrylamide gel
electrophoresis (SOS-PAGE). Gels should be run for 10 hours at a
constant voltage (50V), fixed either stained with silver or with 50
mM (beta-D-glucosyl), Yariv phenylglycoside [(beta-D-Glc), Yariv
reagent in 1% NaCl overnight. Cut relevant fraction of the 9 kD
lily exudate protein and extract the protein fraction. Use Sephadex
0200 column performing fractional collection with the appropriate
buffer. Concentrate the 9 kD lily exudate protein fraction using
Speed-Vac.RTM. system.
3) Preparation of Alginate-N-Oxy-Succinimide:
[0169] 1.20 gm (10.4 mmol) N-Hydroxy-succinimide (NHS) and 10.0
mmol (xgm) Alginic Acid were dissolved in 25 ml of dry DMF in a dry
Argon atmosphere. After addition of 2.26 g (11.0 mmol) dicyclohexyl
carbodiimide (DCC) the solution was stirred overnight at room
temperature. The precipitated solid was filtered off and the filter
rinsed with 5 ml DMF or 5 ml Acetonitrile-Acetone(1:1). Next, the
filtrate was diluted with 180 ml of a mixture of petroleum ether
and isopropanol (6:1) and after 2 hours at room temperature, a
crude product was recovered by filtration, re-dissolved in 5 ml DMF
and recrystallized from Petroleum ether:isopropanol (6:1) to yield
2.8 mmol, 28% product as a white solid after drying in vacuuo.
[0170] The conjugates should be generally synthesized in 10
microliter reactions containing 2.4 mM of the Water lily 9 kD
exudate protein analogue, 0.1M of the respective N-Hydroxy
Succinimide activated Alginate, 0.4M K.sub.2HPO.sub.4 (pH 8.0) and
20% DMF at room temperature. Alter 2-3 hours the reactions should
be quenched with 2 microliter ammonium acetate (1 M) for 30 min.
diluted to 100 microliter. Dialysis should then be performed.
Reconstitution or mixing of the Alginate-9 kD protein conjugate
with lily exudate for the other component responsible for adherence
for forming and evaluation of the Hydrogel should be performed and
Adhesion properties of Lilly Pollen in Vitro should be
performed.
4) Attempt of Conjugation Coupling Reaction Between the Purified 9
kD Lily Protein from Exudate and N-Hydroxy Succinimide Activated
Alginate
[0171] The conjugates should be generally synthesized in 10
microliter reactions containing 2.4 mM of the Water Lily 9 Id)
exudate protein analogue. 0.1M of the respective
N-Hydroxy-Succinimide activated Alginate 0.4M K.sub.2HPO.sub.4 (pH
8.0) and 20% DMF at room temperature. After 2-3 hours the reactions
should be quenched with 2 microliter ammonium acetate (1M) for 30
min. diluted to 100 microliter Dialysis should then be performed
Reconstitution or mixing of the Alginate-9 kD protein conjugate
with Lily exudate for the other component responsible for the
adherence for forming and evaluation of the Target Hydrogel should
be performed and Adhesion properties of Lilly Pollen in Vitro
should be performed.
5) In Vitro Adhesion Assay of Lily Pollen
[0172] Pollen from different anthers suspended in 20 mL of
germination medium, which resulted in an estimated
125,000.+-.17,000 pollen particles. Several 28-mm.sup.2 Petri
dishes were coated with a thin layer (hydrogel layer) of Alginate-9
kD protein (stigma/stylar cysteine-rich adhesin (SCA)) conjugate
from Lily exudates (experimental sample) and non-modified alginate
(control sample), and the 20 mL of pollen suspension was then
added. After short incubation of 10-15 min, the pollen containing
medium aspirated and the alginate films washed by fresh germination
medium (supplemented with calcium chloride 0.05M) three times at
least. The actual number of pollen particles that adhered to the
28-mm.sup.2 surface of Petri dish coated with experimental and
control polymer gels was counted with a stereomicroscope after the
polymer gel film was stained with Coomassie blue (protein
staining).
6) In Vitro Binding Assays Between SCA Protein and Alginate
[0173] Stigma/stylar cysteine-rich adhesin isolated from the stigma
exudate (SCA [SE]) (5 mg) and 50 mg of a carbohydrate polymer
(alginic acid) were combined at pH 6.0 or 10.0. After 15 min of
incubation at room temperature, the mixture (55 mL) was diluted
with 1 mL of 100 mM Tris-HCl buffer, pH 6.0 or 10.0, and passed
through a Centricon filter (molecular mass cutoff of 100 kD;
Millipore, Bedford, Mass.). After being rinsed four times with 1 mL
of buffer each, the retentate and the filtrate were collected and
concentrated by vacuum centrifugation. The dry residues (retentates
and filtrates) were resuspended with an identical volume of SDS
buffer (50 mL) to allow quantitative comparisons, and 10 mL of each
suspension was loaded on 12.5% SDS polyacrylamide gels. For a
control, SCA (5 mg) only was passed through the 1004(13
Centrieonfilter. The interaction pattern between SCA protein and
alginate is analyzed from the SDS-poyacryamide gel results.
* * * * *