U.S. patent application number 10/254430 was filed with the patent office on 2003-02-13 for edta and other chelators with or without antifungal antimicrobial agents for the prevention and treatment of fungal infections.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Hachem, Ray, Raad, Issam, Sherertz, Robert.
Application Number | 20030032605 10/254430 |
Document ID | / |
Family ID | 22007701 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032605 |
Kind Code |
A1 |
Raad, Issam ; et
al. |
February 13, 2003 |
EDTA and other chelators with or without antifungal antimicrobial
agents for the prevention and treatment of fungal infections
Abstract
A pharmaceutical composition comprising at least one antifungal
agent and at least one chelator, and a method for administering the
pharmaceutical composition to a patient having a fungal infection.
Another aspect provides a pharmaceutical composition comprising at
least one chelator, at least one antifungal agent and at least one
monoclonal antibody, wherein the monoclonal antibody is operatively
attached to the chelator, and a method of administering this
composition to a patient having a fungal infection.
Inventors: |
Raad, Issam; (Houston,
TX) ; Sherertz, Robert; (Winston-Salem, NC) ;
Hachem, Ray; (Houston, TX) |
Correspondence
Address: |
David L. Parker
FULBRIGHT & JAWORSKI L.L.P.
600 Congress Avenue, Suite 2400
Austin
TX
78701
US
|
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
22007701 |
Appl. No.: |
10/254430 |
Filed: |
September 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10254430 |
Sep 25, 2002 |
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09680061 |
Oct 4, 2000 |
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09680061 |
Oct 4, 2000 |
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09139522 |
Aug 25, 1998 |
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6165484 |
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60056970 |
Aug 26, 1997 |
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Current U.S.
Class: |
514/28 ;
514/566 |
Current CPC
Class: |
A61K 31/195 20130101;
A61K 31/7048 20130101; A61K 47/183 20130101; A61K 45/06 20130101;
A61K 9/0019 20130101; A61K 31/70 20130101; A61K 45/06 20130101;
A61K 31/70 20130101; A61K 31/195 20130101 |
Class at
Publication: |
514/28 ;
514/566 |
International
Class: |
A61K 031/7048; A61K
031/195 |
Claims
What is claimed is:
1. A method of treating a systemic fungal infection comprising: (a)
obtaining a therapeutically effective amount of a pharmaceutical
composition comprising at least one chelator, at least one
antifungal agent and a pharmaceutical excipient, diluent or
adjuvant; and (b) administering said pharmaceutical composition to
a patient having a fungal infection.
2. The method of claim 1, wherein said chelator in said
pharmaceutical composition may be selected from the group of
chelators in Table 1.
3. The method of claim 2, wherein said chelator is EDTA.
4. The method of claim 1, wherein said antifungal agent may be
selected from the group of antifungal agents in Table 2.
5. The method of claim 4, wherein said antifungal agent is
Amphotericin B.
6. The method of claim 1, wherein said pharmaceutical composition
comprises about 0.001 mg to about 1000 mg of chelator.
7. The method of claim 1, wherein said pharmaceutical composition
comprises about 0.001 mg to about 1000 mg of antifungal agent.
8. The method of claim 1, wherein said pharmaceutical composition
may be administered by injection, bronchoalveoloar lavage, or by
nasal drops, nasal spray, or nasal inhaler.
9. The method of claim 1, wherein said pharmaceutical composition
may be administered by injection.
10. A pharmaceutical composition comprising at least one antifungal
agent and at least one chelator.
11. The pharmaceutical composition of claim 10, wherein the
chelator may be selected from the group of chelators in Table
1.
12. The pharmaceutical composition of claim 10, wherein the
antifungal agent may be selected from the group of antifungal
agents in Table 2.
13. The pharmaceutical composition of claim 11, wherein the
chelator is EDTA.
14. The pharmaceutical composition of claim 12, wherein the
antifungal agent is Amphotericin B.
15. The pharmaceutical composition of claim 10, wherein the
chelator is EDTA and the antifungal agent is Amphotericin B.
16. The pharmaceutical composition of claim 10, further defined as
comprising about 0.001 mg to about 1000 mg of chelator.
17. The pharmaceutical composition of claim 10, further defined as
comprising about 0.001 mg to about 1000 mg of antifungal agent.
18. The pharmaceutical composition of claim 10, further comprising
at least one monoclonal antibody specific for a targeted species of
fungus.
19. The pharmaceutical composition of claim 10, wherein said
monoclonal antibody is operatively attached to said chelator.
20. A pharmaceutical composition comprising at least one chelator,
at least one antifungal agent and at least one monoclonal antibody,
wherein said monoclonal antibody is operatively attached to said
chelator.
21. The pharmaceutical composition of claim 20, wherein the
chelator may be selected from the group of chelators in Table
1.
22. The pharmaceutical composition of claim 20, wherein the
antifungal agent may be selected from the group of antifungal
agents in Table 2.
23. The pharmaceutical composition of claim 21, wherein the
chelator is EDTA.
24. The pharmaceutical composition of claim 22, wherein the
antifungal agent is Amphotericin B.
25. The pharmaceutical composition of claim 20, wherein the
chelator is EDTA and the antifungal agent is Amphotericin B.
26. The pharmaceutical composition of claim 20, further defined as
comprising about 0.001 mg to about 1000 mg of chelator.
27. The pharmaceutical composition of claim 20, further defined as
comprising about 0.001 mg to about 1000 mg of antifungal agent.
28. A method for treating a systemic fungal infection comprising
administering to a patient a therapeutically effective amount of a
pharmaceutical composition comprising at least one chelator, at
least one antifungal agent and at least one monoclonal antibody,
wherein said monoclonal antibody is operatively attached to said
chelator.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/056,970, filed Aug. 26, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the treatment of
fungal infections in mammals. More particularly, the present
invention provides methods of treating fungal infections in mammals
using pharmaceutical preparations including chelator(s), antifungal
agents, and/or monoclonal antibodies. The invention further
provides pharmaceutical compositions useful for treating fungal
infections.
[0004] 2. Description of Related Art
[0005] Fungi, particularly species of Candida, Aspergillus, and
Fusarium are a major cause of infection-related mortality in
patients with leukemia and lymphoma. In addition, fungal infection
is a major cause of mortality in patients with congenital and
acquired deficiencies of the immune system.
[0006] For example, several species of Aspergillus are known to
cause invasive sinopulmonary infections in seriously
immunocompromised patients. Following inhalation of spores,
clinical aspergillosis can occur in three major presentations. The
first presentation, allergic bronchopulmonary aspergillosis,
develops when Aspergillus species colonize the bronchial tree and
release antigens that cause a hypersensitivity pneumonitis. The
second presentation, aspergilloma or "fungus ball," develops in
pulmonary cavities, often in concert with other lung diseases such
as tuberculosis. The third form, invasive pulmonary or disseminated
aspergillosis, is a life threatening infection with a high
mortality rate.
[0007] The drug of choice in treatment of invasive aspergillosis,
as well as in most other systemic mycoses, is Amphotericin B.
Amphotericin B is a polyene antibiotic produced from a strain of
Streptomyces nodosus. It is a lipophilic compound which binds to
ergosterols in fungal membranes, resulting in the formation of
transmembrane channels which allow the escape of metabolites
essential to maintaining the viability of the fungal cell.
Mammalian cell membranes also contain sterols, and it is believed
that this same mechanism of action is responsible for the damaging
effects which Amphotericin B is known to exert on mammalian kidney,
hematopoietic and central nervous system tissues.
[0008] Amphotericin B is not soluble in aqueous solution, and for
this reason it is supplied commercially in the form of a colloidal
suspension comprising Amphotericin B, desoxycholate, and buffers
suspended in a glucose solution. This suspension is usually
administered to the patient intravenously over a period of from two
to six hours; faster infusions can result in cardiorespiratory
arrest. Other possible untoward effects of administering
Amphotericin B include fever, nausea and vomiting, diarrhea, renal
dysfunction, anemia, hypotension, headache, vertigo, and loss of
hearing. Amphotericin B is also available in the form of a
phospholipid complex (ABELCET.RTM., e.g.), which offers the
advantage of somewhat reduced toxicity for those patients who do
not tolerate free Amphotericin B well, although many of the same
untoward side effects may be observed in patients receiving this
lipid complex form of the drug.
[0009] As a consequence of the potential seriousness of its toxic
side effects, there is a clear need for an alternative to treating
systemic mycoses solely with Amphotericin B and/or other harsh
antifungal agents.
SUMMARY OF THE INVENTION
[0010] The present invention provides an effective method of
treating a systemic fungal infection comprising the steps of
obtaining a therapeutically effective amount of a pharmaceutical
composition comprising at least one chelator, at least one
antifungal agent and a pharmaceutical excipient, diluent or
adjuvant, and administering said pharmaceutical composition to a
patient having a fungal infection.
[0011] For the purposes of this disclosure, the phrase
"therapeutically effective amount" is defined as a dosage
sufficient to induce a fungicidal or fungistatic effect upon fungi
contacted by the composition. That amount of the pharmaceutical
composition which is therapeutically effective will depend upon the
ingredients comprising the composition, as well as the treatment
goals.
[0012] For the purposes of this disclosure, the phrase "a chelator"
denotes one or more chelators. As used herein, the term "chelator"
is defined as a molecule comprising nonmetal atoms, two or more of
which atoms are capable of linking or binding with a metal ion to
form a heterocyclic ring including the metal ion.
[0013] For the purposes of this disclosure, the phrase "an
antifungal agent" denotes one or more antifungal agents. As used
herein, the term "antifungal agent" is defined as a compound having
either a fungicidal or fungistatic effect upon fungi contacted by
the compound.
[0014] As used herein, the term "fungicidal" is defined to mean
having a destructive killing action upon fungi. As used herein, the
term "fungistatic" is defined to mean having an inhibiting action
upon the growth of fungi.
[0015] As used herein the terms "contact", "contacted", and
"contacting", are used to describe the process by which a
pharmacological agent, e.g., any of the compositions disclosed in
the present invention, comes in direct juxtaposition with the
target cell.
[0016] Preferable chelators for use in the present invention
include, but are not limited to,
ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); the disodium,
trisodium, tetrasodium, dipotassium, tripotassium, dilithium and
diammonium salts of EDTA; the barium, calcium, cobalt, copper,
dysprosium, europium, iron, indium, lanthanum, magnesium,
manganese, nickel, samarium, strontium, and zinc chelates of EDTA;
trans-1,2-diaminocyclohexane-N,N,N',N'-tetraaceticacid monohydrate;
N,N-bis(2-hydroxyethyl)glycine;
1,3-diamino-2-hydroxypropane-N,N,N',N'-te- traacetic acid;
1,3-diaminopropane-N,N,N',N'-tetraacetic acid;
ethylenediamine-N,N'-diacetic acid;
ethylenediamine-N,N'-dipropionic acid dihydrochloride;
ethylenediamine-N,N'-bis(methylenephosphonic acid) hemihydrate;
N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid;
ethylenediamine-N,N,N',N'-tetrakis(methylenephosponic acid);
O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid;
N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid;
1,6-hexamethylenediamine-N,N,N',N'-tetraacetic acid;
N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid;
1,2-diaminopropane-N,N,N',N'-tetraacetic acid; nitrilotriacetic
acid; nitrilotripropionic acid; the trisodium salt of
nitrilotris(methylenephos- phoric acid);
7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo [11,11,11]
pentatriacontane hexahydrobromide; and triethylenetetramine-N,-
N,N',N",N'",N'"-hexaacetic acid. It is contemplated that any
chelator which binds barium, calcium, cerium, cobalt, copper, iron,
magnesium, manganese, nickel, strontium, or zinc will be acceptable
for use in the present invention.
[0017] More preferably, the chelators for use in conjunction with
the present invention may include
ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); the disodium,
trisodium, tetrasodium, dipotassium, tripotassium, dilithium and
diammonium salts of EDTA; 1,3-diamino-2-hydroxypropane-N,N,-
N',N'-tetraacetic acid; 1,3-diaminopropane-N,N,N',N'-tetraacetic
acid; O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic
acid; and 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo
[11,11,11] pentatriacontane hexahydrobromide.
[0018] Most preferably, the chelators for use in the present
invention may include ethylenediamine-N,N,N',N'-tetraacetic acid
(EDTA); the disodium salt of EDTA;
1,3-diaminopropane-N,N,N',N'-tetraacetic acid; and
O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic
acid.
[0019] Many antifungal agents are known to those of skill in the
art and may be useful in the present invention. For example,
antifungal agents contemplated for use in the present invention
include, but are not limited to, new third generation triazoles
such as UK 109,496, (Voriconazole); SCH 56592; ER30346; UK 9746; UK
9751; T 8581; and Flutrimazole; cell wall active cyclic
lipopeptides such as Cilofungin LY121019; LY303366 (Echinocandin);
and L-743872 (Pneumocandin); allylamines such as Terbinafine;
imidazoles such as Omoconazole, Ketoconazole, Terconazole,
Econazole, Itraconazole and Fluconazole; polyenes such as
Amphotericin B, Nystatin, Natamycin, Liposomal Amphotericin B, and
Liposomal Nystatin; and other antifungal agents including
Griseofulvin; BF-796; MTCH 24; BTG-137586; RMP-7/Amphotericin B;
Pradimicins (MNS 18184); Benanomicin; Ambisome; ABLC; ABCD;
Nikkomycin Z; and Flucytosine.
[0020] More preferably, the antifungal agents for use in
conjunction with the present invention may include polyenes such as
Amphotericin B, Nystatin, Natamycin, Liposomal Amphotericin B, and
Liposomal Nystatin; cell wall active cyclic lipopeptides such as
Cilofungin LY121019; LY303366 (Echinocandin); and L-743872
(Pneumocandin); and other antifungal agents including Griseofulvin
and Flucytosine.
[0021] Most preferably, the antifungal agents for use in the
present invention may include Amphotericin B, Nystatin, Liposomal
Amphotericin B, and Liposomal Nystatin.
[0022] The present invention also provides an effective method of
treating a systemic fungal infection comprising the steps of
obtaining a therapeutically effective amount of a pharmaceutical
composition comprising at least one chelator operatively attached
to a monoclonal antibody, at least one antifungal agent and a
pharmaceutical excipient, diluent or adjuvant, and administering
said pharmaceutical composition to a patient having a fungal
infection. The monoclonal antibody is chosen to bind to a specific
fungal antigen, and may be prepared according to any known method.
The chelators and antifungal agents may be chosen from those
indicated above.
[0023] Monoclonal antibodies useful in conjunction with the present
invention are those that are specific for a targeted species of
fungus. In preferred embodiments, the monoclonal antibodies are
operatively attached to chelators. For the purposes of this
disclosure, the phrase "a monoclonal antibody" denotes one or more
monoclonal antibodies. As used herein, the term "monoclonal
antibody" is defined as an antibody derived from a single clone of
a B lymphocyte. Furthermore, as used herein, the term "operatively
attached" connotes a chemical bond, either covalent or ionic,
between the monoclonal antibody and chelator. As used herein, the
term "specific" indicates that a chemical site on the monoclonal
antibody will recognize and bind with a complementary chemical site
on the surface of the cell of at least the fungal pathogen of
interest.
[0024] The pharmaceutical compositions of the invention are
provided to a patient having a fungal infection in an amount
sufficient to exert a fungicidal or fungistatic effect upon fungi
contacted by the composition. It will be understood with benefit of
this disclosure that such dosages may vary considerably according
to the patient, the infection presented by the patient, and the
particular active ingredients comprising the pharmaceutical
composition.
[0025] The antifungal agents of the present invention may be
administered to a patient in an amount ranging from about 0.001
milligrams per kilogram of body weight per day to about 1000 mg per
kg per day, including all intermediate dosages therebetween. It
will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.001, 0.002, 0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,
52, 53, 54, etc.; 100, 101, 102, 103, 104, etc.; 500, 501, 502,
503, etc.; 600, 700, 800, 900, and about 1000 mg per kg per day,
and including all fractional dosages therebetween.
[0026] More preferably, the antifungal agents of the present
invention may be administered to a patient in an amount ranging
from about 0.01 milligrams per kilogram of body weight per day to
about 100 mg per kg per day, including all intermediate dosages
therebetween. It will be readily understood that "intermediate
dosages", in these contexts, means any dosages between the quoted
ranges, such as about 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.;
3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51, 52, 53,
54, etc.; 60, 70, 80, 90, and about 100 mg per kg per day, and
including all fractional dosages therebetween.
[0027] Most preferably, the antifungal agents of the present
invention may be administered to a patient in an amount ranging
from about 0.1 milligrams per kilogram of body weight per day to
about 10 mg per kg per day, including all intermediate dosages
therebetween. It will be readily understood that "intermediate
dosages", in these contexts, means any dosages between the quoted
ranges, such as about 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9 and
about 10 mg per kg per day, and including all fractional dosages
therebetween.
[0028] The chelators of the present invention may be administered
to a patient in an amount ranging from about 0.001 milligrams per
kilogram of body weight per day to about 1000 mg per kg per day,
including all intermediate dosages therebetween. It will be readily
understood that "intermediate dosages", in these contexts, means
any dosages between the quoted ranges, such as about 0.001, 0.002,
0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6,
7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100,
101, 102, 103, 104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800,
900, and about 1000 mg per kg per day, and including all fractional
dosages therebetween.
[0029] More preferably the chelators of the present invention may
be administered to a patient in an amount ranging from about 0.01
milligrams per kilogram of body weight per day to about 100 mg per
kg per day, including all intermediate dosages therebetween. It
will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7,
8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 60, 70,
80, 90, and about 100 mg per kg per day, and including all
fractional dosages therebetween.
[0030] Most preferably the chelators of the present invention may
be administered to a patient in an amount ranging from about 0.1
milligrams per kilogram of body weight per day to about 10 mg per
kg per day, including all intermediate dosages therebetween. It
will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, and about 10 mg per
kg per day, and including all fractional dosages therebetween.
[0031] The monoclonal antibodies operatively attached to chelators
may be administered to a patient in an amount ranging from about
0.001 milligrams per kilogram of body weight per day to about 1000
mg per kg per day, including all intermediate dosages therebetween.
It will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.001, 0.002, 0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,
52, 53, 54, etc.; 100, 101, 102, 103, 104, etc.; 500, 501, 502,
503, etc.; 600, 700, 800, 900, and about 1000 mg per kg per day,
and including all fractional dosages therebetween.
[0032] More preferably, the monoclonal antibodies operatively
attached to chelators may be administered to a patient in an amount
ranging from about 0.01 milligrams per kilogram of body weight per
day to about 100 mg per kg per day, including all intermediate
dosages therebetween. It will be readily understood that
"intermediate dosages", in these contexts, means any dosages
between the quoted ranges, such as about 0.01, 0.02, 0.03, etc.;
0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14,
etc.; 50, 51, 52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mg
per kg per day, and including all fractional dosages
therebetween.
[0033] Most preferably, the monoclonal antibodies operatively
attached to chelators may be administered to a patient in an amount
ranging from about 0.1 milligrams per kilogram of body weight per
day to about 10 mg per kg per day, including all intermediate
dosages therebetween. It will be readily understood that
"intermediate dosages", in these contexts, means any dosages
between the quoted ranges, such as about 0.1. 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4,
5, 6, 7, 8, 9, and about 10, and including all fractional dosages
therebetween.
[0034] The pharmaceutical compositions of the present invention may
be administered by any known route, including parenterally and
otherwise. This includes oral, nasal (via nasal spray or nasal
inhaler), buccal, rectal, vaginal or topical administration.
Administration may also be by orthotopic, intradermal subcutaneous,
intramuscular, intraperitoneal or intravenous injection and/or
infusion. Such compositions may be administered as pharmaceutically
acceptable compositions that include pharmacologically acceptable
carriers, buffers or other excipients. The phrase
"pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to a human. For treatment of
conditions of the lungs, the preferred route is aerosol delivery to
the lung via bronchoalveolar lavage or the like.
[0035] When administration of the pharmaceutical compositions of
the present invention via intravenous injection and/or infusion is
the preferred route, the pharmaceutical compositions of the present
invention should administered gradually over a period of time
ranging from 0.001 h to 100 h. More preferably, when administration
of the pharmaceutical compositions of the present invention via
intravenous injection and/or infusion is the preferred route, the
pharmaceutical compositions of the present invention should
administered gradually over a period of time ranging from 0.1 h to
50 h. Most preferably, when administration of the pharmaceutical
compositions of the present invention via intravenous injection
and/or infusion is the preferred route, the pharmaceutical
compositions of the present invention should administered gradually
over a period of time ranging from 1 h to 10 h.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein. FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6,
FIG. 7, FIG. 8, FIG. 9, FIG. 10 and FIG. 11 display plots of
microbial population vs. time for cultures of species of
Aspergillus, Candida and Fusarium. Response of these cultures to
treatment with antimicrobials, chelators, and combinations thereof
are indicated.
[0037] FIG. 1 shows the inhibitory effect of EDTA on Aspergillus
flavus in vitro.
[0038] FIG. 2 shows the inhibitory effect of EDTA on Aspergillus
terreus in vitro.
[0039] FIG. 3 shows the inhibitory effect of EDTA on Fusarium
oxysporum in vitro.
[0040] FIG. 4 shows the inhibitory effect of EDTA on Candida krusei
in vitro.
[0041] FIG. 5 shows the synergistic inhibition of Aspergillus
fumigatus by Amphotericin B and EDTA (1.0 mg/mL) in vitro.
[0042] FIG. 6 shows the synergistic inhibition of Aspergillus
fumigatus by Amphotericin B and EDTA (0.1 mg/mL) in vitro.
[0043] FIG. 7 shows the synergistic inhibition of Aspergillus
flavus by Amphotericin B and EDTA (1.0 mg/mL) in vitro.
[0044] FIG. 8 shows the synergistic inhibition of Aspergillus
flavus by Amphotericin B and EDTA (0.1 mg/mL) in vitro.
[0045] FIG. 9 shows the synergistic inhibition of Fusarium solani
by Amphotericin B and EDTA in vitro.
[0046] FIG. 10 shows the synergistic inhibition of Aspergillus
fumigatus by Ambisome and EDTA (0.1 mg/mL) in vitro.
[0047] FIG. 11 shows the synergistic inhibition of Fusarium solani
by Ambisome and EDTA in vitro.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0048] I. Fungal Infections
[0049] Infection is believed to be the cause of death in almost
half of all patients who die of lymphoma, and in almost three
quarters of patients who die of leukemia. Although bacteria are the
causative organisms of many such infections, fungi also play a
major role in these infection-related mortalities. As noted in the
Background section, species of Candida, Aspergillus, and Fusarium
are the major causes of fungal infection-related deaths in patients
with leukemia and lymphoma. Additionally, fungal infection is a
major cause of mortality in patients with congenital and acquired
deficiencies of the immune system.
[0050] In their usual role as saprophytes on decaying vegetation,
Aspergillus organisms are not contagious. Infection arises when
spores of these ubiquitous fungi are aerosolized in the abiotic
environment. No underlying predisposition of race, age, or sex
leads to the development of Aspergillus infection.
[0051] Although Aspergillus infection can be acquired in the
community, the largest threat to the immunocompromised patient is
exposure to contaminated air in the hospital environment. Patients
who have undergone open heart surgery, who have undergone organ or
bone marrow transplantation, or who have prolonged neutropenia
after anticancer chemotherapy may acquire life-threatening
infection either on the wards where they are housed (domiciliary
exposure) or when they are taken to the operating theater,
radiology suite, or catheterization laboratory for an essential
procedure (nondomiciliary exposure). Construction in the hospital,
which results in the liberation of large numbers of spores into the
immediate environment, is particularly hazardous for these patients
(Rubin, 1994; Wade, 1994). Although there is considerable genetic
heterogeneity among Aspergillus strains in nature, newly developed
molecular typing systems have shown that patients are usually
infected with a single strain, a finding that allows the clinician
to rapidly assess whether two or more cases are from the same
environmental source (Birch et al., 1995).
[0052] Several species of Aspergillus are known to cause invasive
sinopulmonary infections in seriously immunocompromised patients.
Following inhalation of spores, clinical aspergillosis can occur in
three major presentations. The first presentation, allergic
bronchopulmonary aspergillosis, develops when Aspergillus species
colonize the bronchial tree and release antigens that cause a
hypersensitivity pneumonitis. The second presentation, aspergilloma
or "fungus ball," develops in pulmonary cavities, often in concert
with other lung diseases such as tuberculosis. The third form,
invasive pulmonary or disseminated aspergillosis, is a life
threatening infection with a very high mortality rate.
[0053] The present invention provides pharmaceutical compositions
and methods for the prevention and treatment of disseminated fungal
infections. It is contemplated that the preparations of the
invention will be useful in eliminating or inhibiting all types of
fungal infections, providing so-called fungicidal or fungistatic
effects. For example, the inventors have discovered that chelators
have significant growth inhibitory effect against species of
Aspergillus (see data in FIG. 1, FIG. 2, FIG. 3 and FIG. 4). The
inventors have further demonstrated conclusively and unexpectedly
that, when combined with antifungal agents, chelators show additive
to synergistic inhibitory activity against the growth of fungal
pathogens (see data in FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG.
10 and FIG. 11). These discoveries provide the basis for a program
of prevention and treatment of systemic fungal infections using any
of several embodiments of the inventive pharmaceutical
formulations, which may comprise various combinations of chelators,
antifungal agents, and any necessary excipients, diluents or
adjuvants.
[0054] In another aspect, the pharmaceutical formulations provided
herein comprise chelators chemically bound, covalently, ionically
or otherwise, to monoclonal antibodies which bind to the fungal
antigen to be treated. Advantageously, the monoclonal antibodies
may serve to deliver the attached chelators directly to their
intended fungal targets, where the chelators may then bind trace
metals such as iron and calcium which might otherwise serve as
fungal virulence factors. The inventors contemplate that this
preferred embodiment even more effectively eliminates or inhibits
fungal infections by decreasing the tendency of the chelators to
bind trace metals with which they may come into contact outside of
the immediate vicinity of the infection
[0055] II. Chelators
[0056] A chelate is the type of coordination compound in which a
central metal ion is attached by coordinate links to two or more
nonmetal atoms in the same molecule. Heterocyclic rings are thus
formed during chelation, with the metal atom as part of the ring.
The molecule comprising the nonmetal linking atoms is termed a
chelator. Chelators are used in various chemical applications, for
example as titrating agents or as metal ion scavengers. Chelators
can be used to remove ions from participation in biological
reactions. For example, the well-known chelator
ethylenediamine-N,N,N',N',-tetraacetic acid (EDTA) acts as an
anticoagulant because it is capable of scavenging calcium ions from
the blood.
[0057] It is known that iron and other trace metals are essential
in the life cycle of microorganisms such as fungi. Without these
trace metals, fungi are unable to grow and reproduce. Although iron
is abundant in nature, its availability for microbial assimilation
is limited owing to the insolubility of ferric ions at neutral or
alkaline pH. As a consequence, many fungi have evolved their own
specialized trace metal-scavenging molecules, called siderophores,
which bind with trace metals and make them available for uptake by
the fungi. The chelators used in conjunction with the present
invention provide an inhibitory effect upon fungal pathogens by
competing with the fungal siderophores for any available trace
metal ions. In this way, the chelators present in the
pharmaceutical preparations of the invention "steal" the metal ions
essential for fungal growth, effectively causing the fungus to
"starve to death." The added antifungal agents and/or monoclonal
antibodies of the preparations of the invention can then come in
and attack the weakened fungi, thereby destroying them or
inhibiting their growth.
[0058] The inventors have discovered that the chelators of the
present invention have significant growth inhibitory effect against
species of Aspergillus. Referring to FIG. 1, it will be seen that
EDTA exerts an inhibitory effect upon Aspergillus flavus relative
to the control population. This effect is most clearly noticeable
beginning 12 h after application of the chelator. Referring to FIG.
2 and FIG. 3, similar inhibitory behavior was noticed in cultures
of Aspergillus terreus and Fusarium oxysporum following application
of EDTA. The inhibitory effect of EDTA on Candida krusei is
noticeable only a few hours after contact of the fungus with the
chelator, as shown by FIG. 4.
[0059] Table 1 provides a representative list of chelators useful
in conjunction with the present invention. The list provided in
Table 1 is not meant to be exhaustive. Preferred chelators are
those which bind trace metal ions with a binding constant ranging
from 10.sup.1 to 10.sup.100; more preferred chelators are those
which bind trace metal ions with a binding constant ranging from
10.sup.10 to 10.sup.80; most preferred chelators are those which
bind trace metal ions with a binding constant ranging from
10.sup.15 to 10.sup.60. Also, preferred chelators are those which
are readily attached to a monoclonal antibody, for example
1,3-diaminopropane-N,N,N',N'-tetraacetic acid (DTPA). Furthermore,
preferred chelators are those which have been shown to have an
inhibitory effect upon target fungal pathogens, for example the
disodium salt of EDTA.
1TABLE 1 CHELATORS ABBREVIATION FULL NAME EDTA free acid
Ethylenediamine-N,N,N',N',-tetraacetic acid EDTA 2Na
Ethylenediamine-N,N,N',N',-tetraacetic acid, disodium salt,
dihydrate EDTA 3Na Ethylenediamine-N,N,N',N',-tetra- acetic acid,
trisodium salt, trihydrate EDTA 4Na
Ethylenediamine-N,N,N',N'-tetraacetic acid, tetrasodium salt,
tetrahydrate EDTA 2K Ethylenefisminr-N,N,N',N'-tetraacetic acid,
dipotassium salt, dihydrate EDTA 2Li Ethylenediamine-N,N,N',-
N'-tetraacetic acid, dilithium salt, monhydrate EDTA 2NH.sub.4
Ethylenediamine-N,N,N',N'-tetraacetic acid, diammonium salt EDTA 3K
Ethylenediamine-N,N,N',N'-tetraacetic acid, tripotassium salt,
dihydrate Ba(II) -EDTA Ethylenediamine-N,N,N',N- '-tetraacetic
acid, barium chelate Ca(II) -EDTA
Ethylenediamine-N,N,N',N'-tetraacetic acid, calcium chelate Ce(III)
-EDTA Ethylenediamine-N,N,N',N'-tetraacetic acid, cerium chelate
Co(II) -EDTA Ethylenediamine-N,N,N',N'-tetraacetic acid, cobalt
chelate Cu(II) -EDTA Ethylenediamine-N,N,N',N'-tetraa- cetic acid,
copper chelate Dy(III) -EDTA Ethylenediamine-N,N,N',N'-tetraacetic
acid, dysprosium chelate Eu(III) -EDTA
Ethylenediamine-N,N,N',N'-tetraacetic acid, europium chelate
Fe(III) -EDTA Ethylenediamine-N,N,N',N'-tetraacet- ic acid, iron
chelate In(III) -EDTA Ethylenediamine-N,N,N',- N'-tetraacetic acid,
indium chelate La(III) -EDTA Ethylenediamine-N,N,N',N'-tetraacetic
acid, lanthanum chelate Mg(II) -EDTA
Ethylenediamine-N,N,N',N'-tetraacetic acid, magnesium chelate
Mn(II) -EDTA Ethylenediamine-N,N,N',N'-tetraacet- ic acid,
manganese chelate Ni(II) -EDTA
Ethylenediamine-N,N,N',N'-tetraacetic acid, nickel chelate Sm(III)
-EDTA Ethylenediamine-N,N,N',N'-tetraacetic acid, samarium chelate
Sr(II) -EDTA Ethylenediamine-N,N,N',N'-tetraaceti- c acid,
strontium chelate Zn(II) -EDTA
Ethylenediamine-N,N,N',N'-tetraacetic acid, zinc chelate CyDTA
trans-1,2-Diaminocyclohexane-N,N,N',N'- tetraaceticacid monohydrate
DHEG N,N-Bis(2-hydroxyethyl)glycine DTPA-OH
1,3-Diamino-2-hydroxypropane-N,N,N',N'- tetraacetic acid DTPA
1,3-Diaminopropane-N,N,N',N'- tetraacetic acid EDDA
Ethylenediamine-N,N'-diacetic acid EDDP Ethylenediamine-N,N'-dipro-
pionic acid dihydrochloride EDDPO Ethylenediamine-N,N'-bis
(methylenephosphonic acid), hemihydrate EDTA-OH
N-(2-Hydroxyethyl)ethylenediamine-N,N',N'- triacetic acid EDTPO
Ethylenediamine-N,N,N',N'-tetrakis (methylenephosponic acid) EGTA
O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'- tetraacetic acid
HBED N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N- diacetic acid
HDTA 1,6-Hexamethylenediamine-N,N,N',N'- tetraacetic acid HIDA
N-(2-Hydroxyethyl)iminodiacetic acid IDA Iminodiacetic acid
Methyl-EDTA 1,2-Diaminopropane-N,N,N',N'- -tetraacetic acid NTA
Nitrilotriacetic acid NTP Nitrilotripropionic acid NTPO
Nitrilotris(methylenephosphoric acid), trisodium salt O-Bistren
7,19,30-Trioxa-1,4,10,13,16,22,27,- 33- octaazabicyclo [11,11,11]
pentatriacontane, hexahydrobromide TTHA Triethylenetetramine -
N,N,N',N",N''',N'''- hexaacetic acid
[0060] III. Monoclonal Antibodies
[0061] Monoclonal antibodies are antibodies derived from a single
clone of B lymphocytes. As such, they are homogenous in structure
and antigen specificity, making them useful as vectors for
directing radionuclides, drugs, or toxins to tissues of interest
such as malignant cells.
[0062] In one embodiment, the pharmaceutical formulations of the
invention may comprise chelators chemically attached to monoclonal
antibodies which have been designed to bind to the fungal antigen
site of interest. Advantageously, the monoclonal antibodies may
serve to deliver the attached chelators directly to their intended
fungal targets, where the chelators may then bind trace metals
which exist in the vicinity of the fungal cell. By delivering the
chelator directly to its intended target, scavenging of any nearby
trace metals which might otherwise serve as fungal virulence
factors is assured.
[0063] Means for preparing and characterizing antibodies are well
known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988; incorporated herein by reference).
The methods for generating monoclonal antibodies (MAbs) generally
begin along the same lines as those for preparing polyclonal
antibodies. Briefly, a polyclonal antibody is prepared by
immunizing an animal with an immunogenic composition and collecting
antisera from that immunized animal. A wide range of animal species
can be used for the production of antisera. Typically the animal
used for production of anti-antisera is a rabbit, a mouse, a rat, a
hamster, a guinea pig or a goat. Because of the relatively large
blood volume of rabbits, a rabbit is a preferred choice for
production of polyclonal antibodies.
[0064] As is well known in the art, a given composition may vary in
its immunogenicity. It is often necessary therefore to boost the
host immune system, as may be achieved by coupling a peptide or
polypeptide immunogen to a carrier. Exemplary and preferred
carriers are keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin can also be used as carriers. Means
for conjugating a polypeptide to a carrier protein are well known
in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hy-
droxysuccinimide ester, carbodiimide and bis-diazotized
benzidine.
[0065] As is also well known in the art, the immunogenicity of a
particular immunogen composition can be enhanced by the use of
non-specific stimulators of the immune response, known as
adjuvants. Exemplary and preferred adjuvants include complete
Freund's adjuvant (a non-specific stimulator of the immune response
containing killed Mycobacterium tuberculosis), incomplete Freund's
adjuvants and aluminum hydroxide adjuvant.
[0066] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal). The production of
polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points following immunization. A
second, booster injection, may also be given. The process of
boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the
immunized animal can be bled and the serum isolated and stored,
and/or the animal can be used to generate MAbs.
[0067] MAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with a selected immunogen
composition, e.g., a purified or partially purified administration
of approximately 10.sup.6 Aspergillus fumigatus conidia (or other
species, such as Aspergillus flavus, Aspergillus terreus, Candida
krusei, or Fusarium solani, or any other fungal pathogen known to
affect humans) in 1.0 mL of sterile NaCl solution with 1.0 mL of
Freund's incomplete adjuvant. The immunizing composition is
administered in a manner effective to stimulate antibody producing
cells. Rodents such as mice and rats are preferred animals,
however, the use of rabbit, sheep frog cells is also possible. The
use of rats may provide certain advantages (Goding, 1986, pp.
60-61), but mice are preferred, with the BALB/c mouse being most
preferred as this is most routinely used and generally gives a
higher percentage of stable fusions.
[0068] Following immunization, somatic cells with the potential for
producing antibodies, specifically B lymphocytes (B cells), are
selected for use in the MAb generating protocol. These cells may be
obtained from biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast
stage, and the latter because peripheral blood is easily
accessible. Often, a panel of animals will have been immunized and
the spleen of animal with the highest antibody titer will be
removed and the spleen lymphocytes obtained by homogenizing the
spleen with a syringe. Typically, a spleen from an immunized mouse
contains approximately 5.times.10.sup.7 to 2.times.10.sup.8
lymphocytes.
[0069] The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell,
generally one of the same species as the animal that was immunized.
Myeloma cell lines suited for use in hybridoma-producing fusion
procedures preferably are non-antibody-producing, have high fusion
efficiency, and enzyme deficiencies that render then incapable of
growing in certain selective media which support the growth of only
the desired fused cells (hybridomas).
[0070] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, pp. 65-66, 1986;
Campbell, pp. 75-83, 1984). For example, where the immunized animal
is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1,
Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;
for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and
U-266, GM 1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in
connection with human cell fusions.
[0071] One preferred murine myeloma cell is the NS-1 myeloma cell
line (also termed P3-NS-1-Ag4-1), which is readily available from
the NIGMS Human Genetic Mutant Cell Repository by requesting cell
line repository number GM3573. Another mouse myeloma cell line that
may be used is the 8-azaguanine-resistant mouse murine myeloma
SP2/0 non-producer cell line.
[0072] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 2:1 proportion, though the
proportion may vary from about 20:1 to about 1:1, respectively, in
the presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Sendai
virus have been described by Kohler and Milstein (1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by
Gefter et al. (1977). The use of electrically induced fusion
methods is also appropriate (Goding pp. 71-74, 1986).
[0073] Fusion procedures usually produce viable hybrids at low
frequencies, about 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are
differentiated from the parental, unfused cells (particularly the
unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0074] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B cells can operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B cells.
[0075] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, plaque assays, dot immunobinding assays, and the like.
[0076] The selected hybridomas would then be serially diluted and
cloned into individual antibody-producing cell lines, which clones
can then be propagated indefinitely to provide MAbs. The cell lines
may be exploited for MAb production in two basic ways. A sample of
the hybridoma can be injected (often into the peritoneal cavity)
into a histocompatible animal of the type that was used to provide
the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific monoclonal antibody
produced by the fused cell hybrid. The body fluids of the animal,
such as serum or ascites fluid, can then be tapped to provide MAbs
in high concentration. The individual cell lines could also be
cultured in vitro, where the MAbs are naturally secreted into the
culture medium from which they can be readily obtained in high
concentrations. MAbs produced by either means may be further
purified, if desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity
chromatography.
[0077] The inventors also contemplate the use of a molecular
cloning approach to generate monoclonals. For this, combinatorial
immunoglobulin phagemid libraries are prepared from RNA isolated
from the spleen of the immunized animal, and phagemids expressing
appropriate antibodies are selected by panning using cells
expressing the antigen and control cells. The advantages of this
approach over conventional hybridoma techniques are that
approximately 10.sup.4 times as many antibodies can be produced and
screened in a single round, and that new specificities are
generated by H and L chain combination which further increases the
chance of finding appropriate antibodies.
[0078] IV. Attachment of Chelators to Monoclonal Antibodies
[0079] Methods for attachment of chelators to proteins, including
monoclonal antibodies, are well known in the art. One of the
earliest efforts at attachment of metal-binding groups to proteins
was that of Gelewitz et al. (1954) who coupled azo-phenanthroline
and azo-oxine to albumin; attempts were made to analyze the
products by colorimetric titration with iron. Later, Sokolovsky et
al., (1967) converted lysozyme tyrosine to 3-aminotyrosine, and
discussed the potential of this procedure to yield new heavy-atom
derivatives for x-ray crystallography. Benisek and Richards (1968)
explored the use of picolinimidate to produce metal-binding sites
on proteins, with a similar goal.
[0080] The first chelate-protein conjugates that were stable enough
for use in vivo were the protein azophenyl-EDTAs of Sundberg et al.
(1974) which resulted from the initial ideas of Baldeschwieler
[Meares et al. (1984)]. Those protein azophenyl-EDTA compounds were
studied over the years 1974-1979, during which the basic
requirements of protein-chelate directed toward in vivo use were
explored (Goodwin et al., 1975; Meares et al., 1976; Leung et al.,
1978; DeRiemer et al., 1979).
[0081] In 1976, Krejcarek and Tucker activated DTPA by a mixed
anhydride method and coupled it to albumin (Krejcarek and Tucker,
1976). The product bound .sup.111In stably enough for many
applications in vivo, and has been used by many nuclear medicine
researchers since (Khaw et al., 1980; Scheinberg, 1982). The
procedure was subsequently improved by Hnatowich et al. (1983).
Comparison of the stability of .sup.111In complexes in human serum
under physiological conditions shows that the indium is lost from
DTPA complexes much more rapidly than from phenyl-EDTA complexes,
whether bound to a protein or not (Yeh et al., 1979). However,
.sup.111In-DTPA complexes decompose slowly enough so that they are
useful in many diagnostic procedures, including those involving
antibodies.
[0082] By 1979, a general method for converting a-amino acids to
bifunctional chelating agents had been devised (Yeh et al., 1979).
This has permitted the synthesis of a wide range of structures from
materials with an interesting choice of useful sidechains.
[0083] Typically, an amino acid such as L-phenylalanine is
nitrated, esterified, and allowed to react with an amine RNH.sub.2.
If R.dbd.H, the final product will be an EDTA analog, whereas if
R.dbd.H.sub.2NCH.sub.2CH- .sub.2--, the final product will be a
DTPA analog. The amide is reduced, and the amines are
carboxymethylated to form a chelating group, and then the aromatic
nitro group is reduced to an amine. This aromatic amine can be
further modified in several ways to form useful derivatives. For
example, treatment with nitric acid renders a diazonium compound
which may react with several different amino acid residues.
[0084] As noted above, in embodiments of the invention where the
chelator is operatively attached to a monoclonal antibody, it is
contemplated that such attachment may be by either covalent or
ionic bond. One example of such an attachment is the diazophenyl
coupling described above, but any other means of chemically binding
a chelator to a monoclonal antibody may be used.
[0085] V. Antifungal Agents
[0086] Fungal infections and the drugs used to treat them have
traditionally been divided into two classes: superficial and
systemic. This distinction is becoming increasingly arbitrary,
however, as some of the drugs previously used to treat only one
class of infection or the other are now used in both cases, with
the differences being ones of mode of administration and/or
concentration of active ingredient. Also, some infections, for
example superficial mycoses, may now be treated either systemically
or topically.
[0087] The classes of drugs used currently to treat systemic fungal
infections include the polyenes, the imidazoles and triazoles,
griseofulvin, and flucytosine. The polyenes bind to ergosterols in
fungal membranes, resulting in the formation of transmembrane
channels which allow the escape of metabolites essential to
maintaining the viability of the fungal cell. Polyenes are highly
toxic. The imidazoles and triazoles are structurally related and
share the same antifungal spectrum and mechanism of action, namely
the inhibition of the fungal sterol 14-.alpha.-demethylase enzyme
system. Griseofulvin was isolated from a species of Penicillium and
acts by inhibiting fungal mitosis. Flucytosine is a fluorinated
pyrimidine which acts upon fungi by inhibiting thymidylate
synthetase.
[0088] In addition to the above well-known classes of antifungal
agents, some compounds more traditionally thought of as
antibacterial agents, for example minocycline (a tetracycline
derivative), have been demonstrated to have a fungistatic or
fungicidal effect on the surface of a venous catheter when
administered in combination with a chelator, for example EDTA, and
other compounds. See, for example, U.S. Pat. No. 5,362,754 by Raad
et al., or U.S. patent application Ser. No. 08/317,309 by Raad et
al., both of which are herein incorporated by reference.
[0089] Antifungal agents particularly preferred in connection with
the present invention include the polyenes, most preferably
Amphotericin B and liposomal Amphotericin B. The inventors have
demonstrated that Amphotericin B acts synergistically in concert
with the chelator EDTA to inhibit species of Aspergillus and
Fusarium. A drug combination is said to exhibit synergism when the
combination achieves a desired effect one order of magnitude or
greater than the analogous effect of the most potent individual
constituent of the combination. For example, referring to FIG. 5,
Amphotericin B at a concentration of 1 .mu.g/mL and EDTA at a
concentration of 1 mg/mL act synergistically to inhibit the growth
of Aspergillus fumigatus by a margin of almost two orders of
magnitude relative to EDTA acting alone. The same effect is
observed when the concentration of EDTA is reduced to 0.1 mg/mL
(FIG. 6). Likewise, Amphotericin B and EDTA inhibit Aspergillus
flavus synergistically, whether EDTA is present at 1.0 mg/mL or 0.1
mg/mL (FIG. 7 and FIG. 8). This synergism extends to inhibition of
Fusarium solani as well, as seen in FIG. 9. FIG. 10 and FIG. 11
show the synergistic inhibitory effect of liposomal Amphotericin B
and EDTA against A. fumigatus and F. solani.
[0090] The inventors have demonstrated, remarkably and for the
first time, that the chelators and antifungal agents of the present
invention act together in a synergistic fashion to inhibit fungal
pathogens. It is contemplated that as a consequence of this
synergism described above between the chelators and the antifungal
agents of the present invention, decreased dosages of antifungal
agent will be sufficient to induce a fungicidal effect in a patient
with a fungal infection, relative to the dosage required when
administering an antifungal agent alone. Advantageously, a
decreased dosage of antifungal agent, when used in conjunction with
the chelators of the present invention, will serve to minimize any
undesirable side effects which antifungal agents may induce in
patients to whom they are administered.
[0091] Table 2 provides a representative list of antifungal agents
useful in conjunction with the present invention. The list provided
in Table 2 is not meant to be exhaustive.
2TABLE 2 ANTIFUNGAL AGENTS UK 109,496 (Voriconazole) Terbinafine
SCH 56592 BF-796 ER30346 MTCH 24 UK 9746 BTG-137586 UK 9751
RMP-7/Amphotericin B T 8581 Omoconazole Flutrimazole Amphotericin B
Cilofungin LY121019 Nystatin LY303366 (Echinocandin) Natamycin
L-743872 (Pneumocandin) Clotrimazole Pradimicins (MNS 18184)
Miconazole Benanomicin Ketoconazole Ambisome Terconazole ABLC
Econazole Liposomal Amphotericin Itraconazole ABCD Fluconazole
Liposomal Nystatin Griseofulvin Nikkomycin Z Flucytosine
[0092] VI. Pharmaceutical Compositions and Routes of
Administration
[0093] Pharmaceutical compositions of the instant invention
comprise an effective amount of at least a chelator dissolved or
dispersed in a pharmaceutically acceptable carrier, such as a
pharmaceutically acceptable buffer, solvent or diluent, or aqueous
medium. Pharmaceutical compositions of the instant invention may
also comprise an effective amount of a chelator and an antifungal
agent dissolved or dispersed in a pharmaceutically acceptable
carrier, such as a pharmaceutically acceptable buffer, solvent or
diluent, or aqueous medium. Additionally, pharmaceutical
compositions of the instant invention may comprise an effective
amount of a chelator operatively attached to a monoclonal antibody
dissolved or dispersed in a pharmaceutically acceptable carrier,
such as a pharmaceutically acceptable buffer, solvent or diluent,
or aqueous medium. Also, pharmaceutical compositions of the instant
invention comprise an effective amount of a chelator operatively
attached to a monoclonal antibody, as well as an antifungal agent,
all dissolved or dispersed in a pharmaceutically acceptable
carrier, such as a pharmaceutically acceptable buffer, solvent or
diluent, or aqueous medium. Such compositions also can be referred
to as inocula.
[0094] The phrases "pharmaceutically acceptable" or
"pharmacologically acceptable" refer to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to a human. As used herein the
terms "pharmaceutically acceptable carrier" and "pharmaceutically
acceptable buffer, solvent or diluent" include any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0095] The therapeutic compositions of the present invention may
include classic pharmaceutical preparations. Administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Alternatively, administration will be by orthotopic,
intradermal subcutaneous, intramuscular, intraperitoneal or
intravenous injection. Such compositions would normally be
administered as pharmaceutically acceptable compositions that
include physiologically acceptable carriers, buffers or other
excipients. For treatment of conditions of the lungs, the preferred
route is aerosol delivery to the lung.
[0096] An effective amount of the therapeutic composition is
determined based on the intended goal. The term "unit dose" or
"dosage" refers to physically discrete units suitable for use in a
subject, each unit containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired
responses, discussed above, in association with its administration,
i.e., the appropriate route and treatment regimen. The quantity to
be administered, both according to number of treatments and unit
dose, depends on the protection desired.
[0097] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting dose include physical and clinical
state of the patient, the route of administration, the intended
goal of treatment (alleviation of symptoms versus cure) and the
potency, stability and toxicity of the particular therapeutic
substance.
[0098] Additional formulations are suitable for oral
administration. Oral formulations include such typical excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. The compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. When the route is topical, the form may be
a cream, ointment, salve or spray.
[0099] As used herein the terms "contact", "contacted", and
"contacting", are used to describe the process by which an
effective amount of a pharmacological agent, e.g., any of the
compounds disclosed in the present invention, comes in direct
juxtaposition with the target cell.
[0100] For methods of treating mammals, pharmaceutical compositions
may be administered by a variety of techniques, such as parenteral,
topical or oral administration. For example, the compositions of
the instant invention may also be formulated for parenteral
administration, e.g., formulated for injection via the intravenous,
intramuscular, sub-cutaneous, or even intraperitoneal routes. The
preparation of an aqueous composition that contains one of the
inventive compounds as an active ingredient will be known to those
of skill in the art in light of the present disclosure. Typically,
such compositions can be prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for use in preparing
solutions or suspensions upon the addition of a liquid prior to
injection can also be employed; and the preparations can also be
emulsified.
[0101] Solutions of the inventive compositions as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0102] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oil
and injectable organic esters such as ethyloleate. Aqueous carriers
include water, alcoholic/aqueous solutions, saline solutions,
parenteral vehicles such as sodium chloride, Ringer's dextrose,
etc. Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial agents, anti-oxidants,
chelating agents and inert gases. The pH and exact concentration of
the various components the pharmaceutical composition are adjusted
according to well known parameters. Sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions may also be useful. In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0103] The compositions of the instant invention may also be
formulated into a composition in a neutral or salt form.
Pharmaceutically acceptable salts include the acid addition salts
(formed, e.g., with any free amino groups present), which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with any free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0104] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0105] Sterile injectable solutions are prepared by incorporating
the compounds of the present invention in the required amount in
the appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0106] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0107] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. In this connection, sterile aqueous media which can be
employed will be known to those of skill in the art in light of the
present disclosure. For example, one dosage could be dissolved in 1
mL of isotonic NaCl solution and either added to 1000 mL of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variations in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject.
[0108] VII. Prevention and Treatment of Disseminated Fungal
Infections
[0109] The pharmaceutical compositions of the invention will be
provided to a patient having a fungal infection in an amount
sufficient to exert a fungicidal or fungistatic effect upon fungi
contacted by the composition. It will be understood with benefit of
this disclosure that such dosages may vary considerably according
to the patient, the infection presented by the patient, and the
particular active ingredients comprising the pharmaceutical
composition.
[0110] The antifungal agents of the present invention may be
administered to a patient in an amount ranging from about 0.001
milligrams per kilogram of body weight per day to about 1000 mg per
kg per day, including all intermediate dosages therebetween. It
will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.001, 0.002, 0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,
52, 53, 54, etc.; 100, 101, 102, 103, 104, etc.; 500, 501, 502,
503, etc.; 600, 700, 800, 900, and about 1000 mg per kg per day,
and including all fractional dosages therebetween.
[0111] More preferably, the antifungal agents of the present
invention may be administered to a patient in an amount ranging
from about 0.01 milligrams per kilogram of body weight per day to
about 100 mg per kg per day, including all intermediate dosages
therebetween. It will be readily understood that "intermediate
dosages", in these contexts, means any dosages between the quoted
ranges, such as about 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.;
3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51, 52, 53,
54, etc.; 60, 70, 80, 90, and about 100 mg per kg per day, and
including all fractional dosages therebetween.
[0112] Most preferably, the antifungal agents of the present
invention may be administered to a patient in an amount ranging
from about 0.1 milligrams per kilogram of body weight per day to
about 10 mg per kg per day, including all intermediate dosages
therebetween. It will be readily understood that "intermediate
dosages", in these contexts, means any dosages between the quoted
ranges, such as about 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9 and
about 10 mg per kg per day, and including all fractional dosages
therebetween.
[0113] The chelators of the present invention may be administered
to a patient in an amount ranging from about 0.001 milligrams per
kilogram of body weight per day to about 1000 mg per kg per day,
including all intermediate dosages therebetween. It will be readily
understood that "intermediate dosages", in these contexts, means
any dosages between the quoted ranges, such as about 0.001, 0.002,
0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6,
7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100,
101, 102, 103, 104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800,
900, and about 1000 mg per kg per day, and including all fractional
dosages therebetween.
[0114] More preferably the chelators of the present invention may
be administered to a patient in an amount ranging from about 0.01
milligrams per kilogram of body weight per day to about 100 mg per
kg per day, including all intermediate dosages therebetween. It
will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7,
8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 60, 70,
80, 90, and about 100 mg per kg per day, and including all
fractional dosages therebetween.
[0115] Most preferably the chelators of the present invention may
be administered to a patient in an amount ranging from about 0.1
milligrams per kilogram of body weight per day to about 10 mg per
kg per day, including all intermediate dosages therebetween. It
will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, and about 10 mg per
kg per day, and including all fractional dosages therebetween.
[0116] The monoclonal antibodies operatively attached to chelators
may be administered to a patient in an amount ranging from about
0.001 milligrams per kilogram of body weight per day to about 1000
mg per kg per day, including all intermediate dosages therebetween.
It will be readily understood that "intermediate dosages", in these
contexts, means any dosages between the quoted ranges, such as
about 0.001, 0.002, 0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,
52, 53, 54, etc.; 100, 101, 102, 103, 104, etc.; 500, 501, 502,
503, etc.; 600, 700, 800, 900, and about 1000 mg per kg per day,
and including all fractional dosages therebetween.
[0117] More preferably, the monoclonal antibodies operatively
attached to chelators may be administered to a patient in an amount
ranging from about 0.01 milligrams per kilogram of body weight per
day to about 100 mg per kg per day, including all intermediate
dosages therebetween. It will be readily understood that
"intermediate dosages", in these contexts, means any dosages
between the quoted ranges, such as about 0.01, 0.02, 0.03, etc.;
0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14,
etc.; 50, 51, 52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mg
per kg per day, and including all fractional dosages
therebetween.
[0118] Most preferably, the monoclonal antibodies operatively
attached to chelators may be administered to a patient in an amount
ranging from about 0.1 milligrams per kilogram of body weight per
day to about 10 mg per kg per day, including all intermediate
dosages therebetween. It will be readily understood that
"intermediate dosages", in these contexts, means any dosages
between the quoted ranges, such as about 0.1. 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4,
5, 6, 7, 8, 9, and about 10 mg per kg per day, and including all
fractional dosages therebetween.
[0119] The pharmaceutical compositions of the present invention may
be administered by any known route, including parenterally and
otherwise. This includes oral, nasal (via nasal spray or nasal
inhaler), buccal, rectal, vaginal or topical administration.
Administration may also be by orthotopic, intradermal subcutaneous,
intramuscular, intraperitoneal or intravenous injection and/or
infusion. Such compositions may be administered as pharmaceutically
acceptable compositions that include pharmacologically acceptable
carriers, buffers or other excipients. The phrase
"pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to a human. For treatment of
conditions of the lungs, the preferred route is aerosol delivery to
the lung via bronchoalveolar lavage or the like.
[0120] VIII. Packaging and Kits
[0121] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe or other
container means, into which the linked antibody/chelator may be
placed, and preferably, suitably aliquoted. Where a second or third
antifungal agent, other chelator, or additional component is
provided, the kit will also generally contain a second, third or
other additional container into which this component may be placed.
The kits of the present invention will also typically include a
means for containing the antibody/chelator, antifungal agent, other
chelator, and any other reagent containers in close confinement for
commercial sale. Such containers may include injection or
blow-molded plastic containers into which the desired vials are
retained.
[0122] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE 1
Synergy Study
[0123] The goal of this study was to determine if there is a
synergistic effect between EDTA and Amphotericin B and EDTA and
Ambisome respectively. The data collected are displayed in FIG. 5,
FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10 and FIG. 11. These figures
are discussed elsewhere in the Detailed Description section. The
studies were conducted in a laboratory incubator at a constant
temperature of 30.degree. C.
[0124] The medium was a single lot of liquid RPMI 1640 medium
(Whittaker Bioproducts, Inc., Walkersville, Md.) supplemented with
0.3 g of L-glutamine per liter and 0.165 M MOPS buffer (34.54
g/liter) and without sodium bicarbonate.
[0125] Test inocula contained approximately 1.times.10.sup.3 to
1.times.10.sup.4 conidia/mL. To induce conidium and sporangiophore
formation, fungi were grown on sabouraud dextrose agar plates at
35.degree. C. for 5 to 7 days. Each fungus was then covered with
approximately 2 mL sterile 0.85% saline water. The suspension was
then harvested by gently probing the colonies with sterile glass
rods. The resulting mixture of conidia or sporangiophores and
hyphal fragments was withdrawn and filtered through a sterile
4.times.4 gauze to a sterile tube. The homogenous suspension was
later mixed with a vortex mixer for 30 s and the densities of the
suspension were read and adjusted to a range of 80 to 85%
transmittance. Inoculum of 0.1 mL was delivered to each flask
containing 20 mL of RPMI and drug dilution series. The final
conidia concentration ranged from 1.times.10.sup.3 to
1.times.10.sup.4 conidia/mL. A control flask was maintained without
any drugs. The flasks were incubated in a shaker at 30.degree. C.
for 24 to 48 h. Glass beads were added to all flasks with visible
fungal growth in an attempt to homogenize the solution and achieve
even distribution of conidia for culture. Cultures were done at 0,
4, 24, and 48 h on sabouraud dextrose agar plates and incubated at
35.degree. C. for 48 h.
[0126] Amphotericin B for injection, USP (Gensia Laboratories,
LTD.) was suspended and diluted in sterile water and stored at 1
mg/mL in a glass vile in the dark at -70.degree. C.
[0127] Ambisome was obtained in 50 mg vials and used immediately
upon opening of the vial. Typically, 50 mg of Ambisome was diluted
in 12 mL of sterile water. Further dilutions were performed as
needed.
[0128] Edetate disodium INJ., USP (Abbott Laboratories, North
Chicago, Ill.) was stored at a concentration of 150 mg/mL at
4.degree. C.
[0129] Further dilutions were made to achieve the desired
concentration of each drug at the time of the study. For
Amphotericin B and Ambisome, the concentration was 1.0 .mu.g/mL and
for EDTA the concentrations were 0.1 and 1.0 mg/mL.
EXAMPLE 2
Inhibition Study
[0130] The goal of this study was to determine if chelators have an
inhibitory effect on species of Aspergillus, Fusarium, and Candida.
The data collected are displayed in FIG. 1, FIG. 2, FIG. 3 and FIG.
4. These figures are discussed elsewhere in the Detailed
Description section. A spectrophotometer was used at a frequency of
660 nm to measure the absorbency of the solution.
[0131] For molds, all inocula were started at 1.times.10.sup.4
conidia/mL. For yeast and bacteria, all inocula were started at
1.times.10.sup.6 cfu/mL. The medium used was Mueller-Hinton.
[0132] The concentration of chelator in these studies was 0.35 mg
EDTA per mL water.
EXAMPLE 3
In Vivo Prophetic Model
[0133] In vivo studies will be conducted with either rabbits or
mice, both of which are suitable animal models. Immunosupression
with cyclophosphamide should be given intravenously 3 days prior to
commencement of the study in order to achieve neutropenia by the
day of animal infection.
[0134] Treatment with all drugs begins 18 to 24 h after infection
and continues for 10 days.
[0135] All animals surviving to day 11 are sacrificed. Their lungs,
kidneys, liver, and spleen are removed and transferred into 5 mL of
sterile saline, homogenized in a tissue grinder for 15 to 30 sec,
and diluted to 10.sup.-1, 10.sup.-2, 10.sup.-3, and 10.sup.-4. A
total of 1.0 mL of each dilution is spread onto a sabouraud
dextrose agar plate and allowed to grow by incubating them at
37.degree. C. The plates are then counted for quantitative
analysis. Also, histopathology analysis will be conducted on all
organs analyzed.
[0136] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
REFERENCES
[0137] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference.
[0138] Benisek and Richards, J. Biol. Chem., 243:4267, 1968.
[0139] Campbell, In: Monoclonal Antibody Technology, Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 13, Burden
and Von Knippenberg, (Eds.), Amsterdam, Elseview,pp 75-83, 1984
[0140] DeRiemer, Meares, Goodwin, Diamanti, J. Med Chem., 22:019,
1979.
[0141] Gefter et al., Somatic Cell Genet., 3:231-236, 1977.
[0142] Gelewitz, Riedemann, Klotz, Arch. Biochem. Biophys., 53:411,
1954.
[0143] Goding, 1986, In: Monoclonal Antibodies: Principles and
Practice, 2nd ed., Academic Press, Orlando, Fla., pp. 60-61, 65-66,
71-74, 1986.
[0144] Goodwin, Sundberg, Diamanti, Meares, In:
Radiopharmaceuticals, Society of Nuclear Medicine, New York, p. 80,
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