Note: Descriptions are shown in the official language in which they were submitted.
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
PSEUDOMYCIN ANTIFUNGAL COMPOSITIONS
AND METHODS FOR THEIR USE
FIELD OF THE INVENTION
The present invention relates to methods and compositions for treating fungal
infections that include formulations of a pseudomycin or a
lipodepsidecapeptide and a
cyclodextrin, in particular, hydroxypropyl-(3-cyclodextrin or sulfobutylether-
~i-
cyclodextrin.
BACKGROUND
Fungal infections are a significant cause of disease, degradation of quality
of life,
and mortality among humans, particularly for immune compromised patients. The
incidence in fungal infections in humans has increased greatly in the past 20
years. This
is in part due to increased numbers of people with immune systems weakened or
devastated by organ transplants, cancer chemotherapy, AIDS, age, and other
similar
disorders or conditions. Such patients are prone to attack by fungal pathogens
that are
prevalent throughout the population but are kept in check by a functioning
immune
system. These pathogens are difficult to control because some existing
antifungal agents
are either highly toxic or only inhibit fungal activity. For example, the
polyenes are
fungicidal but toxic; whereas, the azoles are much less toxic but only
fungistatic. More
importantly, there have been recent reports of azole and polyene resistant
strains of
Candida which severely limits therapy options against such strains.
One class of new antifungal agents, the pseudomycins, shows great promise for
treating fungal infections in a variety of patients. Pseudomycins are natural
products
derived from isolates of Pseudomonas syringae. P. syringae is a large family
of plant
bacteria that have been the source of several bioactive substances, such as
bacitracin and
the syringomycins. Natural strains and transposon generated mutants of P.
syringae
produce compounds with antifungal activity. A transposon generated regulatory
mutant
of the wild type strain of P. syringae 174, known as MSU 16H (ATCC 67028),
produces
several pseudomycins. For example, pseudomycins A, B, C and C' have each been
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
isolated and purified and their structures have been characterized by methods
including
amino acid sequencing, NMR, and mass spectrometry and have been shown to
possess
wide spectrum antifungal activity, including activity against important fungal
pathogens
in both humans and plants. See, e.g. Ballio et al., "Novel bioactive
lipodepsipeptides
from Pseudomonas syringae: the pseudomycins," FEBS L.ett. 355, 96-100 (1994)
and
U.S. Patent No. 5,576,298. The pseudomycins, the syringomycins, the
syringotoxins, and
the syringostatins represent structurally distinct families of antifungal
compounds.
As yet, formulations of pseudomycins and related antifungal agents from P.
syringae that are effective for antifungal therapy have not been developed.
Typically
injection of a pseudomycin is accompanied by unacceptable adverse side effects
such as
irntation at the injection site. There remains a need for a composition of a
pseudomycin
or a related antifungal agent that can be used for treating fungal infections
without the
adverse side effects observed in certain formulations of pseudomycins.
SUMMARY OF THE INVENTION
The present invention relates to a method and composition effective for
administration of a pseudomycin or a related antifungal agent to a patient in
need thereof.
The method includes treating a fungal infection by administering an effective
amount of a
composition including a pseudomycin, such as pseudomycin B, or a related
antifungal
agent and hydroxypropyl-(3-cyclodextrin or sulfobutylether-(3-cyclodextrin.
The method
is effective to reduce symptoms of the fungal infection and, preferably, kills
the fungus
causing the infection. The method is effective against infections of fungi
including
Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Histoplasma
capsa~latum. In a second embodiment, the method reduces adverse effects of
injecting a
pseudomycin, such as pseudomycin B, or a related antifungal agent by
administering the
pseudomycin or a related antifungal agent in a composition including
hydroxypropyl-~i-
cyclodextrin or sulfobutylether-~3-cyclodextrin. In another embodiment, the
method
includes administering toxicologically relevant doses of a pseudomycin, such
as
pseudomycin B, or a related antifungal agent in a composition including the
pseudomycin
and hydroxypropyl-(3-cyclodextrin or sulfobutylether-(3-cyclodextrin.
2
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
The composition of the method includes a pseudomycin or related antifungal
agent, such as pseudomycin B, or a related antifungal agent and hydroxypropyl-
(3-
cyclodextrin or sulfobutylether-(3-cyclodextrin. Such a composition is
effective for
treating or reducing symptoms of fungal infections, for reducing adverse
effects of
injecting a pseudomycin, such as pseudomycin B, or a related antifungal agent
and for
administering toxicologically relevant doses of a pseudomycin, such as
pseudomycin B,
or a related antifungal agent. The composition includes the cyclodextrin in a
molar excess
over the pseudomycin or a related antifungal agent, preferably in at least
about a two-fold
molar excess and is suitable for parenteral administration. The composition
can include
excipients such as sodium chloride, mannitol, or dextrose which adjust the
toxicity of the
composition. The compositions containing a pseudomycin or a
lipodepsidecapeptide
antifungal agent, or a pharmaceutically acceptable salt, hydrate or ester
thereof, in
combination with a hydroxyalkyl-~i-cyclodextrin or a sulfoalkylether-~i-
cyclodextrin may
be used for the manufacture of a medicament for the treatment of a fungal
infection.
Definitions
As used herein, the term "pseudomycin" refers to compounds having formula I:
O
HO
O
OH
O~ N H
NH H N OH
HO, O
NH ~CI
O
O O
H2N
NH
O O O 'NHR
N NH
O
OH NH2
H2N O
3
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
where R is a lipophilic moiety. The lipophilic moiety includes C9-Ci5 alkyl,
C9-C~5
hydroxyalkyl, Cg-C,5 dihydroxyalkyl, C9-C,5 alkenyl, C9-C15 hydroxyalkenyl, or
C9-C15
dihydroxyalkenyl. The pseudomycin compounds A, A', B, B', C, C' are
represented by
the formula I above where R is as defined below.
Pseudomycin A R = 3,4-dihydroxytetradecanoyl ..
Pseudomycin A' R = 3,4-dihydroxypentadecanoate
Pseudomycin B R = 3-hydroxytetradecanoyl
Pseudomycin B' R = 3-hydroxydodecanoate
Pseudomycin C R = 3,4-dihydroxyhexadecanoyl
Pseudomycin C' R = 3-hydroxyhexadecanoyl
As used herein, the term "lipodepsidecapeptide" refers to compounds having
formula II:
HO\
NFiz
II
where R is a lipophilic moiety. The lipophilic moiety includes C9-CIS alkyl,
C9-C,;
hydroxyalkyl, C9-C,5 dihydroxyalkyl, C~-C,; alkenyl, C9-C15 hydroxyalkenyl, or
C9-Ci5
dihydroxyalkenyl. Preferably, the lipophilic moiety is C" alkyl. The alkyl,
hydroxyalkyl,
dihydroxyalkyl, alkenyl, hydroxyalkenyl, or dihydroxyalkenyl groups may be
branched or
4
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
unbranched. Preferably, the amino acid sequence of the depsidecapeptide ring
is
threonine-alanine-threonine-glutamine-homoserine-dehydroaminobutyric acid-
alanine-
dehydroalanine-threonine-arginine, referred to herein as "25-B 1 decapeptide"
or
"Thr-Ala-Thr-Gln-Xaa-Xaa-Ala-Xaa-Thr-Arg (SEQ ID NO: 1 )".
As used herein, the term "25-B 1 decapeptide antifungal agent A" refers to the
specific
depsidecapeptide having the preferred amino acid sequence SEQ 1D NO: 1 and R
is an
unbranched C" alkyl (i.e., R = -(CHZ),oCH3).
Any derivatives or analogs of the above described pseudomycins and related-
pseudomycins (e.g., lipodepsidecapeptides) are also considered to be within
the spirit of
the present invention. Although a specific conformation is depicted above,
other
stereoisomers and diastereoisomers are also contemplated to be within the
spirit of the
present invention.
DETAILED DESCRIPTION
Pseudomycins and Related Antifun. ag 1 Agents
As used herein, pseudomycin (PS) refers to one or more members of a family of
antifungal agents that has been isolated from the bacterium Pseudomonas
syringae. A
pseudomycin is a lipodepsipeptide, a cyclic peptide including one or more
unusual amino
acids and having one or more appended hydrophobic or fatty acid side chains.
Specifically, the pseudomycins are lipodepsinanopeptides, with a cyclic
peptide portion
closed by a lactone bond and including the unusual amino acids 4-
chlorothreonine
(ClThr), 3-hydroxyaspartic acid (HOAsp), 2,3-dehydro-2-aminobutyric acid
(Dhb), and
2,4-diaminobutyric acid (Dab). It is believed that these unusual amino acids
are involved
in biological characteristics of the pseudomycins, such as stability in serum
and their
killing action. Pseudomycins include pseudomycin A, pseudomycin A',
pseudomycin B
(PSB), pseudomycin B', pseudomycin C, and pseudomycin C'. Each of these
pseudomycins has the same cyclic peptide nucleus, but they differ in the
hydrophobic side
chain attached to this nucleus.
Pseudomycins A, A', B, B', C and C' have each been isolated and purified and
their structures have been characterized by methods including sequencing, NMR,
and
5
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
mass spectrometry. See, e.g., U.S. Patent No. 5,576,298, issued November 19,
1996 to
Strobel et al. Harrison et al.); "Pseudomycins, a family of novel peptides
from
Pseudomonas Syringae possessing broad-spectrum antifungal activity" J. Gen.
Microbiology 137, 2857-2865 (1991); Ballio et al., "Novel bioactive
lipodepsipeptides.
from Pseudomonas syringae: the pseudomycins" FEBS Lett. 355, 96-100 (1994);
and
Coiro, V.M., et al., "Solution conformation of the Pseudomonas syringae MSU
16H
phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations
using
distance geometry and molecular dynamics from NMR data," Eur. J. Biochem.,
257(2),
449-456 (1998). Antifungal activity due to pseudomjrcins was traced to P.
syringae
bearing a transposon known as Tn 903, which encodes kanamycin resistance. The
sequence of and methods for manipulating transposon Tn 903 are known. Oka et
al. J.
Mol. Biol. 147, 217-226 (1981); "Nucleotide sequence of the kanamycin
resistance
transposon Tn 903." Methods for growth of various strains of P. syringae and
their use in
production of antifungal agents such as pseudomycins are disclosed in U.S.
Patent
Application Serial No. PCT/LJS00/08728 by Matthew D. Hilton, et al. entitled
"Pseudomycin Production By Pseudomonas Syringae" submitted evendate herewith
and
described below for the production of the pseudomycins and
lipodepsidecapeptides from
biological materials on deposit (see Examples). Pseudomycins A' and B' are
described in
U.S. Patent Application Serial No. PCT/US00/08727, by Palaniappan Kulanthaivel
et al.
entitled " Pseudomycin Natural Products" submitted evendate herewith and may
be
produced using the general procedures described below. Each of the references
cited in
this paragraph is specifically incorporated herein by reference.
The pseudomycins vary in structure and properties. The pseudomycins exhibit
activity against a wide variety of fungi and also exhibit generally acceptable
toxicity.
Compared to the other pseudomycins, pseudomycin B has greater potency against
certain
fungi and a lower level of toxicity. Therefore, for the present formulations
and methods.
pseudomycin B is preferred. The structure of pseudomycin B is represented by
Formula I
above where R =
3-hydroxytetradecanoyl. Pseudomycin B has a cyclic nonapeptide ring having the
sequence Ser-Dab-Asp-Lys-Dab-Thr-Dhb-HOAsp-ClThr, more specifically, L-Ser-D-
6
RECTLFIED SHEET (PJLE 91)
lSA/ EP
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-O~-L-Thr(4-CI) with the carboxyl
group of the CIThr and the hydroxyl group of the serine closing the ring with
a lactone
bond. The amine group of the serine forms an amide bond with the carboxyl of a
3-
monohydroxytetradecanoyl moiety. The carboxyl group of the serine forms an
amide
bond with the Dab of the ring.
As used herein, antifungal agents related to pseudomycins include
lipodepsidecapeptide antifungal agents produced by Pseudomonas syringae. A
representative of this class of compounds, 25-B 1 decapeptide antifungal agent
A, has been
purified and its structure determined. Lipodepsidecapeptide antifungal agents,
such as 25-
B 1 decapeptide antifungal agent A, are described in U.S. Patent Application
Serial No.
PCT/LJS00/08724, by Palaniappan Kulanthaivel et al. entitled "Antifungal
Agents Isolated
From Pseudomonas Syringae" submitted evendate herewith and may be produced
using
the general procedures described below. This patent application is
specifically
incorporated herein by reference.
Method for Pseudomycin Production
To produce one or more pseudomycins from a wild type or mutant strain of P.
syringae, the organism is cultured with agitation in an aqueous nutrient
medium including
an effective amount of three or fewer amino acids. The three or fewer amino
acids are
preferably glutamic acid, glycine, histidine, or a combination thereof. In one
preferred
embodiment, the amino acids include glycine and, optionally, one or more of a
potato
product and a lipid. Culturing is conducted under conditions effective for
growth of P.
syringae and production of the desired pseudomycin or pseudomycins. Effective
conditions include temperature of about 22°C to about 27°C, and
a duration of about 36
hours to about 96 hours. When cultivated on the media such as those described
herein, P.
syringae can grow in cell densities up to about 10-15 g/L dry weight and
produce
pseudomycins in total amounts at least about 10 p,g/mL, preferably at least
about 40
~g/mL, more preferably about 500 p,g/mL or more.
Controlling the concentration of oxygen in the medium during culturing of P.
syringae is advantageous for production of a pseudomycin. Preferably, oxygen
levels are
7
RECTIFIED SHEET (RULE 91)
ISA / EP
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
maintained at about 5% to about 50% saturation, more preferably about 30%
saturation.
Sparging with air, with pure oxygen, or with gas mixtures including oxygen can
regulate
the concentration of oxygen in the medium. Further, adjustment of the
agitation rate can
be used to adjust the oxygen transfer rate.
Controlling the pH of the medium during culturing of P. syringae is
advantageous
for production of a pseudomycin. Pseudomycins are labile at basic pH, and
significant
degradation can occur if the pH of the culture medium is above about 6 for
more than
about 12 hours. Preferably, the pH of the culture medium is maintained at less
than about
6, more preferably less than about 5.5, and most preferably above 4Ø The pH
is
preferably maintained at about 5 to about 5.4, more preferably about 5.0 to
about 5.2.
Although not limiting to the present invention, it is believed that
pseudomycin
degradation at basic pH is due to opening of the lactone ring and leaving of
Cl-.
P. syringae can produce one or more pseudomycins when grown in batch culture.
However, fed-batch or semi-continuous feed of glucose and, optionally, an acid
or base,
such as ammonium hydroxide, to control pH, enhances pseudomycin production.
Pseudomycin production by P. syringae can be further enhanced by using
continuous feed
methods in which glucose and, optionally, an acid or base, such as ammonium
hydroxide,
to control pH, are fed automatically.
Employing P. syringae, one or more pseudomycins can be produced in substantial
quantities. That is, total quantities of one or more pseudomycins from at
least about 200
p,g/mL to about 900 ~,g/mL, preferably of from about 600 p,g/mL to about 900
~g/mL,
more preferably from about 800 ~g/mL to about 900 pg/mL. Preferably, for
production of
pseudomycin A, pseudomycin A is produced in total quantities from at least
about 10
p,g/mL to about 400 pg/mL, more preferably of from about 300 ~g/mL to about
400
~g/mL, most preferably from about 350 p,g/mL to about 400 ~g/mL. Preferably,
for
production of pseudomycin B, pseudomycin B is produced in total quantities
from at least
about 10 p.g/mL to about 300 ~g/mL, more preferably from about 200 ~g/mL to
about 300
~tg/mL, most preferably from about 250 ~tg/mL to about 300 ~tg/mL. Preferably,
for
production of pseudomycin C, pseudomycin C is produced in total quantities
from at least
about 5 p.g/mL to about 100 ~JmL, more preferably of from about 5 ~g/mL to
about 50
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
~g/mL, most preferably from about 10 ~,g/mL to about 50 pg/mL. Preferably, for
production of pseudomycin C', pseudomycin C' is produced in total quantities
from at
least about 1 p,g/mL to about 50 pg/mL, more preferably of from about 10
p,g/mL to about
50 pg/mL, most preferably from about 30 ~g/mL to about 50 p.g/mL. Effective
conditions
for the production of Pseudomycin A' and/or B' include temperature of about
22°C to
about 27°C, and a duration of about 36 hours to about 96 hours. When
cultivated on the
media such as those described herein, P. syringae can grow in cell densities
up to about
10-15 g/L dry weight and produce pseudomycins A' and/or B' in total amounts at
least
about 10 p,g/mL.
Choice of P. syringae strain can affect the amount and distribution of
pseudomycin or pseudomycins produced by culturing under the conditions
described
herein. For example, strains MSU 16 H and 67 H1, and like strains, each
produce
predominantly pseudomycin A, but also produce pseudomycin B and C, typically
in ratios
of roughly 4:2:1. Strain 67 H1 and like strains, however, typically produce
levels of
pseudomycins about 3- to about 5-fold larger than are produced by strain MSU
16H.
Compared to strains MSU 16 H and 67 Hl, strain 25-B 1 and like strains produce
more
pseudomycin B and less pseudomycin C. Strain 7H9-1 and like strains are
distinctive in
producing predominantly pseudomycin B and for producing larger amounts of
pseudomycin B than other strains. For example this strain can produce
pseudomycin B in
at least a 10-fold excess over either pseudomycin A or C. Pseudomycin A'
and/or B' can
be produced from mutants of P. syringae. The mutants are produced by treating
the
bacteria with an amount of a mutagenic agent effective to produce mutants that
overproduce pseudomycin A' and/or B', that produce pseudomycin A' and/or B' in
excess
over other pseudomycins, or that produce pseudomycin A' and/or B' under
advantageous
growth conditions. While the type and amount of mutagenic agent to be used can
vary, a
preferred method is to serially dilute NTG to levels ranging from 1 to 100
~Cg/ml. The
mutants overproduce pseudomycin A' and/or B' preferably to at least about 10
~,g/mL.
Each pseudomycin or mixtures of pseudomycins can be detected, determined,
isolated, and/or purified by any of a variety of methods known to those of
skill in the art.
For example, the level of pseudomycin activity in a broth or in an isolated or
purified
9
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
composition can be determined by antifungal action against a fungus such as
Candida.
Numerous methods are known for the preparation and analysis of the
pseudomycins. For
example, one or more pseudomycins can be isolated and purified by
chromatography,
such as HPLC.
Method for Producing a P. syrinQae Lipodepsidecapeptide
To produce one or more P. syringae lipodepsidecapeptides, such as 25-B 1
decapeptide antifungal agent A, from a wild type or mutant strain of P.
syringae, the
organism is cultured with agitation in an aqueous nutrient medium
including an effective amount of three or fewer amino acids. The three or
fewer amino acids are preferably glutamic acid, glycine, histidine, or a
combination thereof. In one preferred embodiment, the amino acids
include glycine and, optionally, one or more of a potato product and a
lipid. Culturing is conducted under conditions effective for growth of P.
syringae and
production of a desired P. syringae lipodepsidecapeptide, such as 25-B 1
decapeptide
antifungal agent A. Effective conditions include a temperature of about
22° C to about
27° C, and a duration of about 36 hours to about 96 hours. When
cultivated on media
such as those described herein, P. syringae can grow at cell densities up to
about 10-15
g/L dry weight and produce a P. syringae lipodepsidecapeptide, such as 25-B 1
decapeptide antifungal agent A, in a total amount at least about 10 ~,glmL,
preferably at
least about 50 ~.g/mL.
Controlling the concentration of oxygen in the medium during culturing of P.
syringae is advantageous for production of a P. syringae lipodepsidecapeptide,
such as
25-Bl decapeptide antifungal agent A. Preferably, oxygen levels are maintained
at about
5% to about 50% saturation, more preferably about 30% saturation. Sparging
with air,
with pure oxygen, or with gas mixtures including oxygen can regulate the
concentration
of oxygen in the medium. Further, adjustment of the agitation rate can be used
to adjust
the oxygen transfer rate.
Controlling the pH of the medium during culturing of P. syringae is
advantageous
for production of a P. syringae lipodepsidecapeptide, such as 25-B 1
decapeptide
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
antifungal agent A. The pH of the culture medium can be maintained at less
than about 6
and above about 4.
P. syringae can produce a P. syringae lipodepsidecapeptide, such as 25-B 1
decapeptide antifungal agent A, when grown in batch culture. However, fed-
batch or
semi-continuous feed of glucose and, optionally, an acid or base, such as
ammonium
hydroxide, to control pH, enhances production of a P. syringae
lipodepsidecapeptide,
such as 25-B 1 decapeptide antifungal agent A. Production of a P. syringae
lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, by P.
syringae can
be further enhanced by using continuous culture methods in which glucose and,
optionally, an acid or base, such as ammonium hydroxide, to control pH, are
fed
automatically. The pH is preferably maintained at a pH of about 5 to about
5.4, more
preferably about 5.0 to about 5.2.
Choice of P. syringae strain can affect the amount and distribution of a P.
syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A,
produced
by culturing under the conditions described herein. For example, strain 25 B 1
can
produce predominantly 25-B 1 decapeptide antifungal agent A.
The cyclic decapeptide nucleus of the P. syringae lipodepsidecapeptides can be
prepared by cleaving off the lipophilic moiety, such as by deacylation.
Cleavage and
deacylation methods are well-known to those skilled in the art, such as the
use of
deacylase enzymes.
Cyclodextrins
As used herein, "cyclodextrin" refers to a compound including cyclic alpha(1--
~4)
linked D-glucopyranose units. Typically, cyclodextrins are formed by the
action of an
amylase on starch. a-cyclodextrin refers to a cyclodextrin with 6 cyclic,
linked D-
glucopyranose units, ~i-cyclodextrin has 7 cyclic, linked D-glucopyranose
units, and ~y-
cyclodextrin has 8 cyclic, linked D-glucopyranose units. These cyclic, linked
D-
glucopyranose units define a hydrophobic cavity, and cyclodextrins are known
to form
inclusion compounds with other organic molecules, with salts, and with
halogens either in
the solid state or in aqueous solutions. Most natural, unsubstituted
cyclodextrins exhibit
11
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
unacceptable toxicity in pharmaceutical compositions and are not readily
eliminated once
in a patient's blood stream. Methods for preparing cyclodextrins are well-
known to those
skilled in the art.
Cyclodextrins include substituted cyclodextrins. As used herein, "substituted
cyclodextrin" refers to a derivative of a cyclodextrin formed by addition of a
side chain to
one or more positions on the cyclodextrin. The side chain can be an organic
moiety, or a
heteroorganic moiety. Substituted cyclodextrins include cyclodextrins that
have been
alkylated, hydroxy alkylated or reacted to form a sulfoalkyl ether.
Substituted
cyclodextrins include hydroxypropyl cyclodextrin, hydroxyethyl cyclodextrin,
glucosyl
cyclodextrin, maltosyl cyclodextrin, and sulfobutylether cyclodextrin. Methods
for
preparing cyclodextrin derivatives are known in the art, and certain methods
are described
in U.S. Patent Nos. 4,727,064 (issued to Pitha on February 23, 1988) and
5,134,127
(issued to Stella et al. on July 28, 1992), European Patent Application
Publication
Number 0 499 322 A1, and Pitha et al., Int. J. Pharm., 29, 73-82 (1986). The
disclosures
of each of the documents cited in this paragraph is specifically incorporated
herein by
reference.
The cyclodextrins vary in structure and properties. For example, the size
(e.g.
diameter, and depth) and functionality (e.g. hydrophobicity, charge,
reactivity, and ability
to hydrogen bond) of the hydrophobic cavity varies among the substituted and
unsubstituted a-, (3-, and'y-cyclodextrins. Typically, a cyclodextrin selected
for a
formulation has a size and functionality that compliments the other components
of the
formulation. For the present formulations and methods, it is believed that
substituted
cyclodextrins like the hydroxyalkyl cyclodextrins and sulfoalkylether
cyclodextrins have a
size and functionality that compliment the other components of the
formulation, such as
the pseudomycin or related antifungal agent, such as pseudomycin B. Preferred
cyclodextrins include hydroxypropyl-~i-cyclodextrin (HP-CD), and
sulfobutylether-~3-
cyclodextrin (SBE-CD).
Sulfobutylether cyclodextrin is commercially available and can be synthesized
by
methods known in the art. As used herein, sulfobutylether cyclodextrin or SBE-
CD refers
to the sulfobutylether derivative of ~3-cyclodextrin typically including an
average of about
12
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
1 to about 8, preferably about 6 to about 7 sulfobutylether groups at the 2,
3, and 6
positions on the ~3-cyclodextrin ring. Hydroxypropyl cyclodextrin is
commercially
available and can be synthesized by methods known in the art. As used herein,
hydroxypropyl cyclodextrin or HP-CD refers to the 2-hydroxypropyl derivative
of (3-
cyclodextrin typically including an average of about 8 2-hydroxypropyl groups
at 2, 3, and
6 positions on the (3-cyclodextrin ring.
Biological Activities of Pseudomycins and Related Antifunsal Agents
Pseudomycins have several biological activities including killing various
fungi,
such as fungal pathogens of plants and animals. In particular, pseudomycins or
related
antifungal agents are active antimycotic agents against fungi that cause
opportunistic
infections in immune compromised individuals. These fungi include Cryptococcus
neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and various species
of
Candida including C. parapsilosis, C. albicans, C. glabrata, C. tropicalis,
and C. krusei.
Killing, rather than inhibiting the growth of fungi, particularly of fungal
pathogens, is a
desirable and preferred biological activity of a pseudomycin.
The pseudomycins have also been shown to be toxic to a broad range of plant-
pathogenic fungi including Rynchosporium secalis, Ceratocystis ulmi,
Rizoctonia solani,
Sclerotinia sclerotiorum, Verticillium albo-atrum, Verticilliccm dahliae,
Thielaviopis
basicola, Fusarium oxysporum and Fusarium culmorum. (see Harnson, L., et al.,
"Pseudomycins, a family of novel peptides from Pseacdomonas syringae
possessing
broad-spectrum antifungal activity, " J of General Microbiology, 7, 2857-2865
(1991).)
In addition, P. syringae MSU 16H has been shown to confer a greater protection
than the
wild-type strain in elms infected with Ceratocystic ulmi, the causal agent of
Dutch elm
disease. (see e.g., Lam et al, Proc. Natl. Sci. USA, 84, 6447-6451 (1987)).
Pseudomycins or related antifungal agents have certain adverse biological
activities, or adverse effects. As used herein, adverse effects of a
pseudomycin or related
antifungal agent refers to adverse effects occurnng at or near the site of
injection of a
pseudomycin or related antifungal agent. Of particular interest are the
adverse effects of a
pseudomycin, such as pseudomycin B, or a related antifungal agent. For
example,
13
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
intravenous injection of a pseudomycin, such as pseudomycin B, in an acetate
buffer
formulation results in damaging the vein into which the pseudomycin was
injected.
Outside the vein (i.e., extravasation), pseudomycin damages tissue near the
site of
injection. The adverse effects of a pseudomycin, such as pseudomycin B, on the
vein and
on surrounding tissues include destruction of the endothelium of the vein,
destruction of
tissue, inflammation, and local toxicity to host tissues. Although not
limiting to the
present invention, it is believed that intravenous injection of the
pseudomycin results in
loss of vascular endothelium and extravasation of the pseudomycin which, in
turn, results
in adverse effects on the tissues around the vein.
Formulation and Antifun~al Action of a Pseudomycin or Related Antifun~aL 1
Agent
Adverse effects result from injection of numerous different formulations of a
pseudomycin, such as pseudomycin B, or a related antifungal agent including an
acetate
buffer formulation, a saline formulation, a microemulsion formulation, an
emulsion
formulation, a Povidone K12 complex formulation, and a micelle formulation.
The
observed adverse effects with these vehicles prevent administration of
effective antifungal
amounts of the pseudomycin. The adverse effects typically require halting
dosing with
the pseudomycin or related antifungal agent after only one or a few
injections, and before
a sufficient antifungal effect is obtained. Even in their milder forms, such
adverse effects
at the site of injection can discourage patient compliance with an effective
dosing regime.
Therefore, an effective antifungal method employing pseudomycin or related
antifungal
agent requires a formulation that allows administration of repeated doses.
Such a
formulation permits administration of repeated doses of pseudomycin or related
antifungal agent of at least about 0.01 mg/kg, preferably more than about 0.1
mg/kg,
preferably about 0.1 to about 5 mg/kg, more preferably about 0.1 to about 0.5
mg/kg,
without unacceptable adverse effects at the site of injection.
The observed adverse effects also prevent administration of doses of a
pseudomycin, such as pseudomycin B, or a related antifungal agent approaching
the
maximum tolerated dose, that is, the lowest dose that causes significant
systemic toxicity
or target organ toxicity in the host. Such high doses of pseudomycin are
typically about
14
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
0.5 mg/kg, preferably about 5 mg/kg or more, preferably about 5 to about 25-50
mg/kg.
Even a single administration of pseudomycin in vehicles such as an acetate
buffer can
cause adverse effects that prevent further administration of pseudomycin or
related
antifungal agent. Such adverse effects can prevent administration of a
toxicologically
relevant dose of pseudomycin or related antifungal agent, which prevents
determining
relevant toxicological parameters for the antifungal agent. Such high doses
also become
necessary for therapy of fungal infections for strains or organisms tolerant
of or resistant
to a pseudomycin, such as pseudomycin B, or a related antifungal agent.
Formulating a pseudomycin, such as pseudomycin B, or related antifungal agent
with a cyclodextrin, such as HP-CD, SBE-CD, or a like cyclodextrin, allows
effective
antifungal or toxicological dosing of the agent. Such a formulation reduces or
prevents
adverse effects of injection of the pseudomycin or related antifungal agent.
Pseudomycin
B is soluble in aqueous solution to at least about 15 mg/mL, so the
cyclodextrin is not
typically needed to increase the solubility of this antifungal agent.
Pseudomycin B and
HP-CD or SPE-CD can be dissolved without difficulty at suitable concentrations
in an
aqueous vehicle such as saline for injection, or known mannitol or dextrose
vehicles. The
toxicity of the formulation can be adjusted with, for example, NaCI to bring
it within an
appropriate physiological range, if necessary.
Preferably, a formulation of a pseudomycin, such as pseudomycin B, or a
related
antifungal agent with HP-CD, SBE-CD, or a like cyclodextrin includes about
0.05 mg/mL
to about 20 mg/mL of pseudomycin, preferably about 1 to about 15 mg/mL. The
formulation includes HP-CD, SBE-CD, or a like cyclodextrin in an amount
effective to
reduce the adverse effects of injection of pseudomycin or related antifungal
agent and to
allow toxicological or antifungal dosing of this antifungal agent. Typically,
HP-CD, SBE-
CD, or a like cyclodextrin are present in the formulation at about 0.5 wt-%
(wt/vol of
solution) to about 6 wt-%, preferably about 1 wt-% to about 5 wt-%, preferably
about 2
wt-%. Advantageously, HP-CD, SBE-CD, or a like cyclodextrin is present in a
molar
excess over the pseudomycin, such as pseudomycin B, or a related antifungal
agent,
preferably, at least about a 2-fold to about a 4-fold molar excess. Dosing of
such an
effective toxicological or antifungal formulation can occur for at least about
1-5,
CA 02371942 2001-10-11
WO 00/62793 PCT/CTS00/08725
preferably about 2-3 injections per day for about 1-7 days without
unacceptable adverse
effects at the site of injection.
Although not limiting to the present invention, it is believed that the
adverse
effects of pseudomycins or related antifungal agents upon injection are due in
part to
effects of soluble aggregates of the agent. For example, a pseudomycin, such
as
pseudomycin B, or a related antifungal agent includes a peptide ring and a
long fatty chain
and it is believed a long fatty chain may induce the formation of micelles in
aqueous
solutions or in blood. According to this model, it is desirable to reduce the
size of the
soluble aggregates of the pseudomycin or related antifungal agent. This can be
accomplished by manipulating the composition of the vehicle to reduce the
impetus of the
pseudomycin or related antifungal agent to aggregate. Alternatively, the
vehicle can
include an agent that complexes the pseudomycin or related antifungal agent
and prevents
the aggregates from damaging the vein and surrounding tissues. For example, a
complex
of the fatty chain of the pseudomycin or related antifungal agent with a
cyclodextrin or
substituted cyclodextrin, can, according to this model, provide a form of the
pseudomycin
or related antifungal agent that does not cause the adverse reaction at the
site of injection.
When administered in an effective antifungal amount, a composition of a
pseudomycin, such as pseudomycin B, or a related antifungal agent with a
cyclodextrin,
such as HP-CD or SPE-CD, reduces the burden of a fungal infection, reduces
symptoms
associated with the fungal infection, and can result in elimination of the
fungal infection.
The fungal infection can be eliminated because pseudomycins can kill, rather
than just
reducing the growth of fungi.
A typical patient in need of antifungal therapy with a formulation of a
pseudomycin, such as pseudomycin B, or a related antifungal agent and HP-CD,
SBE-CD,
or a like cyclodextrin has severe symptoms of infection, such as high fever,
and is likely
to be in intensive or critical care. Various fungi can cause such serious
infections.
Candida, for example, causes mucosal and serious systemic infections and
exists in
strains that are resistant to azole and polyene antifungals. Aspergillats
causes life-
threatening systemic infections. Cryptococcais is responsible for meningitis.
Such serious
fungal infections may occur in immune compromised patients, such as those
receiving
16
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
organ or bone marrow transplants, undergoing chemotherapy for cancer,
recovering from
major surgery, or suffering from HIV infection.
The antifungal therapy includes administration, typically parenteral
administration,
preferably intravenous administration, of a formulation of a pseudomycin, such
as
pseudomycin B, or a related antifungal agent and HP-CD, SBE-CD, or a like
cyclodextrin
over several days to halt the infection. For most fungal infections reduction
of symptoms
of the infection includes reduction of fever, return to consciousness, and
increased well
being (e.g. the patient acts and feels better) of the patient. Preferably,
symptoms are
reduced by killing the fungus to eliminate the infection or to bring the
infection to level
tolerated by the patient or controlled by the patient's immune system.
Pharmaceutical Compositions Including a Formulation of a Pseudomycin or
Related
Antifungal Agent and a Cyclodextrin
A pharmaceutical composition including a formulation of a pseudomycin, such as
pseudomycin B, or a related antifungal agent and a cyclodextrin, such as SBE-
CD or HP-
CD, can also include carriers, excipients, vehicles, and other additives. The
formulation can
include additives such as various oils, including those of petroleum, animal,
vegetable or
synthetic origin, for example, peanut oil, soybean oil, mineral oil, and
sesame oil. Suitable
pharmaceutical excipients include starch, cellulose, glucose, lactose,
sucrose, gelatin, malt,
magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride,
dried skim
milk, glycerol, propylene glycol, water, and ethanol. The compositions can be
subjected to
conventional pharmaceutical expedients, such as sterilization, and can contain
conventional
pharmaceutical additives, such as preservatives, stabilizing agents, wetting,
or emulsifying
agents, salts for adjusting osmotic pressure, and buffers. Suitable
pharmaceutical Garners
and their formulations are described in Martin, "Remington's Pharmaceutical
Sciences,"
15th Ed.; Mack Publishing Co., Easton (1975); see, e.g., pp. 1405-1412 and pp.
1461-1487.
Such compositions will, in general, contain an effective amount of the active
compound
together with a suitable amount of carrier so as to prepare the proper dosage
form for proper
administration to the host.
17
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Typically, the compositions will be administered to a patient (human or other
animal, including mammals such as, but not limited to, cats, horses and cattle
and avian
species) in need thereof, in an effective amount to inhibit the fungal
replication. For
systemic administration, the daily dosage as employed for adult human
treatment will range
from 5 mg to 5000 mg of active ingredient, preferably 50 mg to 2000 mg, which
can be
administered in 1 to 5 daily doses, for example, depending on the route of
administration
and the condition of the patient. When the compositions include dosage units,
each unit will
preferably contain 2 mg to 2000 mg of active ingredient, for example 50 mg to
500 mg. For
serious infections, the compound can be administered by intravenous infusion
using, for
example, 0.01 to 10 mg/kg/hr of the active ingredient.
Uses of Pseudomycin-Cyclodextrin Formulations
The present invention also encompasses a kit including the present
pharmaceutical
compositions and to be used with the methods of the present invention. The kit
can contain
a vial which contains a formulation of the present invention and suitable
carriers, either
dried or in liquid form. The kit further includes instructions in the form of
a label on the
vial and/or in the form of an insert included in a box in which the vial is
packaged, for the
use and administration of the compounds. The instructions can also be printed
on the box in
which the vial is packaged. The instructions contain information such as
sufficient dosage
and administration information so as to allow a worker in the field to
administer the drug. It
is anticipated that a worker in the field encompasses any doctor, nurse, or
technician who
might administer the drug.
The present invention also relates to a pharmaceutical composition including a
formulation of a pseudomycin, such as pseudomycin B, or a related antifungal
agent and a
cyclodextrin, and that is suitable for administration by injection. According
to the
invention, a formulation of a pseudomycin, such as pseudomycin B, or a related
antifungal agent and a cyclodextrin can be used for manufacturing a
composition a
pseudomycin, such as pseudomycin B, or a related antifungal agent and a
cyclodextrin in
a form that is suitable for administration by injection. For example, a liquid
or solid
formulation can be manufactured in several ways, using conventional
techniques. A
18
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
liquid formulation can be manufactured by dissolving a pseudomycin, such as
pseudomycin B, or a related antifungal agent and cyclodextrin in a suitable
solvent, such
as water, at an appropriate pH, including buffers or other excipients.
Agricultural Compositions Including a Formulation of a Pseudomycin or Related
Antifun~al Agent and a Cyclodextrin
Compositions containing one or more pseudomycin, lipodepsidecapeptide, or
combinations thereof (including hydrates, solvates, and esters thereof) and a
cyclodextrin
(e.g., SBE-CD or HP-CD) may be used in the treatment of fungi in plants (in
particular, V.
albo-atrum, Rhizoctonia solani and F. oxysporum) either as a direct treatment
or
preventative treatment. Generally, the infected plants are treated by
injecting or spraying
an aqueous suspension of the P. syringae antifungal agents into or onto the
plant. Means
of injection are well-known to those skilled in the art (e.g., gouge pistol).
Any means of
spraying the suspension may be used that distributes an effective amount of
the active
material onto the plant surface. The suspension may also include other
additives
generally used by those skilled in the art, such as solubilizers, stabilizers,
wetting agents,
and combinations thereof.
The present invention may be better understood with reference to the following
examples. These examples are intended to be representative of specific
embodiments of
the invention, and are not intended as limiting the scope of the invention.
EXAMPLES
Biological Materials on Deposit
P. syringae MSU 16H is publicly available from the American Type Culture
Collection, Parklawn Drive, Rockville, MD, USA as Accession No. ATCC 67028. P.
syringae strains 25-B1, 7H9-l, and 67 H1 were deposited with the American Type
Culture Collection on March 23, 2000 and were assigned the following Accession
Nos.:
25-Bl Accession No. PTA-1622
7H9-1 Accession No. PTA-1623
67 H1 Accession No. PTA-1621
19
CA 02371942 2001-10-11
WO 00/62793 PCT/L1S00/08725
Example 1 -- Adverse Reaction to Intravenous Infection of a Formulation of
Pseudomvcin B in Saline for Infection and Without Cyclodextrin
Pseudomycin B formulated in saline for injection, without any cyclodextrin,
was
evaluated in a 1-week pilot study for adverse reaction upon intravenous
injection.
Materials and Methods
CD-1 mice (males) were divided into groups of four and were given 0, 1, 5, 10,
25, or 50 mg /kg/day PSB-TFA as an intravenous injection (slow bolus) into a
lateral tail
vein. The vehicle used for this study was saline for injection. The potency of
the
trifluoroacetic acid salt of pseudomycin B (PSB-TFA) was adjusted to account
for the
trifluoroacetic acid present in the salt form. The data collected included
live phase (i.e.,
clinical observations, body weight, and mortality), clinical chemistry,
limited organ
weights, and limited histopathology (kidney, liver, heart, and injection
site).
Results
All animals survived to the scheduled termination on day 7. However, each of
the
mice exhibited a severe adverse reaction at the site of injection. This began
as marked
swelling and darkening of the tail, typically occurnng within about 6 to 8
hours after
injection. The severity of the tissue reaction in all animals receiving a dose
of >_ 10 mg/kg
resulted in discontinuation of dosing after 2 to 5 doses. There were no
significant clinical
chemistry, gross pathology, or histopathology findings observed to indicate
target organ
effects other than evidence of severe adverse reaction at the injection site.
Example 2 --Evaluation of the Adverse Reaction of Animal Cells
to Pseudomvcin B and Related Compounds
Pseudomycin B and related compounds were tested for their in vitro toxicity
toward mammalian cells to evaluate whether toxicity was related to the
structure of the
compound.
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Materials and Methods
Pseudomycin B (PSB) and the related compound pseudomycin A (PSA) were
tested at a concentration of 0.5 mg/mL for their effect on L6 skeletal muscle
cells. This in
vitro muscle model has been demonstrated to correlate with in vivo parenteral
antibiotic
venous irritation. See: Rosalki S.B., An Improved Procedure for Serum Creatine
Phosphokinase Determination. J. Lab. Clin. Chem. 69: 696-705 (1967); D.M.
Hoover et
al., Fundamental and Applied Toxic. 14: 589-597 (1990); Mosmann T., Rapid
Colorimetric Assay for Cellular Growth and Survival, J. Immunol. Method 65: 55-
63
(1983).
Toxicity was determined by monitoring the levels of creatine phosphokinase
(CPK) and MTT reductase activities in the cells by standard methods.
Results
The data are summarized in Table 1 which identifies the compounds tested and
reports the amount of creatine phosphokinase retention (percent of control),
and the MTT
reductase activity (percent of control). The greater the toxicity the lower
the measured
levels of CPK retention and MTT reductase activity.
Table 1 -- In Vitro Toxicity of Pseudomycin B and Related Compounds.
Com ound Descri CPK MTT
tion
PSB-TFA 0 13
PSB-FB 0 10
PSC 0 3
~ PSA 0 11
Conclusion
This in vitro cellular toxicity of pseudomycins A and B, and suggests that
formulation of these compounds may be necessary to reduce adverse reactions
upon
intravenous injection.
21
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Example 3 -- Evaluation of Adverse Reactions and Their Amelioration Upon
Intravenous Infection of Various Formulations of Intravenous Pseudomycin B
These three studies examined the effects of various formulations of
pseudomycin
B on the adverse reactions observed at the site of injection, and amelioration
of the
adverse reaction, upon intravenous injection.
Study 1
This study evaluated formulations including pseudomycin B in an acetate
buffer,
in a microemulsion, and with SBE-CD.
Materials and Methods
Animals were as described above in Example 1. The doses selected were based on
the adverse effects observed in Figure 1. The low dose was 5 mg/kg, which, in
Example
1, caused slight tissue injury but allowed dosing for 7 full days. The high
dose was 25
mg/kg, which, in Example 1, resulted in severe tissue injury and restricted
dosing. This
study tested three formulations acetate buffer, a microemulsion vehicle, and a
sulfobutylether-(3-cyclodextrin (SBE-CD) vehicle at each of these two doses of
PSB-TFA.
The microemulsion vehicle includes 1.7 wt-% propylene glycol, 3.4 wt-%
emulphor EL,
1.4 wt-% phospholipon 90, 3.5 wt-% fractionated coconut oil in O.OSM acetate
buffer
with 1.75 wt-% dextrose, at pH 4.5.
SBE-CD was obtained from Cydek, Inc., 12980 Metcalf Ave., Suite 470, Overland
Park, Kansas 66213 and was prepared as 2 wt-% Sulfobutylether-(3-cyclodextrin
in O.OSM
acetate buffer with 1.75 wt-% dextrose, pH 4.5. Typically a SBE-CD stock
solution is
prepared by mixing glacial acetic acid with deionized water and NaOH to
achieve a
solution that is 0.05 M in acetic acid and at pH 4.5. Solid dextrose and SBE-
CD are
dissolved in the acetic acid buffer to the desired concentration. Then
pseudomycin B is
added to achieve the desired concentration and the solution is stirred until
the
pseudomycin dissolves.
22
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Results
The study design and results are summarized in the Table 2.
Table 2 -- Reactions to Various Formulations of Pseudomycin B (PSB).
Grou Vehicle Dose of Results
PSB
O1 acetate buffer5 m k Received full 7 doses, tail swellin
noted.
02 acetate buffer25 mg/kg Received 2-3 doses, tail swelling
after first dose,
bruising after second dose, and
blackened tails
by day 4. A portion of the tail
from 2 animals
was missin on da S.
03 microemulsion'S mg/kg Received full 7 doses, tails
looked normal
throu hout stud .
04 microemulsion'25 mg/kg Received 2 doses, tail swelling
after first and
second doses. The tails recovered
to the point at
which they could have been dosed
again by day
5 but further recovery was monitored
through the
end of the stud .
OS SBE cyclodextrin'5 mg/kg Received 7 full doses, tails
looked normal
throu hout the stud .
06 SBE cyclodextrin'25 mg/kg Received 7 full doses, tails
looked normal
throu hout the stud .
1. / wt-~/o Propylene glycol, 3.4 wt-% Emulphor EL, 1.4 wt-% Phospholipon 90,
3.5 wt-%
fractionated coconut oil in O.OSM acetate buffer with 1.75 wt-% dextrose, pH
4.5
z 2 wt-% Sulfobutylether-(3-cyclodextrin in O.OSM acetate buffer with 1.75 wt-
% dextrose, pH 4.5
Conclusion
The results of this study indicate that SBE-CD vehicle completely ameliorated
the
adverse effects exhibited by pseudomycin B upon intravenous administration of
doses up
to 25 mg/kg. The microemulsion reduced the adverse effects at both doses.
However, at
the higher doses, the adverse effects were sufficient that dosing had to be
suspended.
Study 2
This study evaluated formulations including pseudomycin B in an acetate
buffer,
with SBE-CD, with HP-CD, with gamma-CD, in an emulsion, in a Povidone
formulation,
and in a micelle formulation.
Materials and Methods
Animals were as described in Example 1 and were used in groups of three.
Pseudomycin B was administered at a dose of 25 mg/kg/day for 7 days by
intravenous
bolus administration into a lateral tail vein at 10 mLlkg. The vehicle for
Group 1
(positive control) was acetate buffer alone since this has been shown (Example
1 and
23
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Example 2, Study 1) to result in marked adverse reactions. The vehicle for
Group 2
(negative control) was acetate buffer containing 2 wt-% SBE-CD which has been
shown
(Example 2, Study 1) to completely ameliorate the adverse effects observed
when
pseudomycin B is administered in acetate buffer. The other vehicles are as
follows:
Group 3, 2 wt-% hydroxypropyl-(3-cyclodextrin; Group 4, 2 wt-% gamma-
cyclodextrin;
Group 5, lyposin II emulsion, which includes 10% soybean oil and 10% safflower
oil;
Group 6, Povidone K12 emulsion, which includes Polyvinylpyrrolidone 2000-3000
m.w.;
and Group 7, 0.5 wt-% polysorbate 80 micelle preparation, which includes
dextrose,
polysorbate 80, pH 4.5 acetate buffer.
Results
The results of the study are shown in Table 3.
Table 3 -- Reactions to Various Formulations of Pseudomycin B.
Treatment Day Day Day Day Day Day Day
1 2 3 3 4 5 6
O.OSM acetate buffer Positive
Control
2 wt-% SBE- CD Ne alive Control
2 wt-% H drox ro 1-CD
2 wt-% Gamma-CD
L osin II emulsion .~'~~~;
Povidone K 12 com lex ;~
0.5 wt-% Pol sorbate 80 micelle ~.~a'~,.~:. ',
re aration
Normal dosing
'~~~v Partial dosing and/or swelling/discoloration noted after dosing
No dosing
Animals in acetate buffer, Povidone, and polysorbate groups exhibited severe
tail vein irritation.
Day 2 - One animal in the Gamma-CD group died due to a dosing accident.
Another animal in
this group exhibited reddening of its tail.
Day 4 - The animal that had tail reddening on Day 2 was found dead due to
causes unknown.
The results of this study confirmed the irntation potential of pseudomycin B
when
administered in acetate buffer. Group 1 showed swelling and discoloration of
the tail
after the first dose which impaired dosing on the second and third day. By day
4, the
adverse reaction prevented further dosing of the animals. SBE-CD prevented the
adverse
reaction observed in Group 1. Group 2 received 7 daily doses of 25 mg/kg
pseudomycin
B with no evidence of adverse reactions. Groups 3 and 4, formulations
including
24
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
hydroxypropyl-(3-cyclodextrin or gamma-cyclodextrin, also showed protection
from the
adverse reactions caused by pseudomycin B. The Liposin II emulsion provided
only
partial protection. Although the animals were dosed daily for 7 days, tail
swelling
impaired dosing on the last 3 days of the study. The Povidone K12 complex and
0.5 wt-
% Polysorbate 80 micelle preparation provided no protection compared with
acetate
buffer alone.
Conclusion
This study showed that additional cyclodextrins, such as hydroxypropyl-(3-
cyclodextrin and gamma-cyclodextrin also exhibit the protective effect.
However, the
results with gamma-CD may be equivocal due to the tail reddening and
unexplained death
seen with one animal in this group.
Study 3
This study evaluated formulations including pseudomycin B in saline for
injection
at two pHs, in pluronic F68, in solutol, and in polysorbate.
Materials and Methods
Animals were female CDl mice handled according to established protocols. The
mice were divided into groups of three. Pseudomycin B was administered at a
dose of 25
mg/kg/day for 7 days by intravenous bolus administration into a lateral tail
vein at 10
mL/kg. The vehicle for Group 2 was 0.9 wt-% NaCI (saline for injection) at pH
4.5. The
vehicle for Group 3 was 0.9 wt-% NaCI (saline for injection) at pH 6.5. The
vehicle for
Group 4 was 0.9 wt-% NaCI in 0.05 wt-% Pluronic F68, which includes propylene
oxide
and ethylene oxide copolymers at various m.w. and HLB at pH 6.5. The vehicle
for Group
5 was 0.9 wt-% NaCI in 0.5 wt-% Pluronic F68 at pH 6.5. The vehicle for Group
6 was
0.9 wt-% NaCI in 0.05 wt-% Solutol, which includes 70% polyglycol ester of 12-
hydroxystearate and 30% polyethylene glycol at pH 6.5. The vehicle for Group 7
was 0.9
wt-% NaCI in 0.05 wt-% polysorbate 20 at pH 6.5.
25
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Results
The results of the study are shown in Table 4. Clinical observations that
affected
all treatment groups included swollen and discolored tails. In addition,
individual animals
in Groups 03-07 demonstrated evidence of tissue necrosis. Three animals died
on study
(Animal 6051 on test day 5 and Animals 2053 and 6052 on test day 6). All of
these
deaths were the result of mechanical injuries sustained during dosing and,
therefore, were
not considered compound-related.
Table 4 -- Reactions to Various Formulations of Pseudomycin B.
AnimalDa Da Da Da 3 Da 4 Da 5 Da 6 Da 7
0 1 2
2051 0 0 0 1 1 1 ~'C11,? ,
~~~~ ~. ~
~ ~
2052_ 0 0 0 1 1 1 1 1
2033 0 0 0 N~~ ~ ",
:= f
rY .~~
3051 1 1 3 1 2 3 ' ~~ ~;2~, L~~~;~
2 2 PD ~3 UB;~~;~ ~ . f
> > > > > - ~
3 ~ ~
~
3052 0 0 1 ~ ~. ~ ~3~ ~ ~;2 3; ~T a . .
; , ~ > r
....
3053 0 1,2 1,2 1,2,3 ~,3> y2,3~~ , . ~.
, w
'
4051 0 1.2 3 =~. = x= r~l~.I 2,3'la
~: : ~ ~ ~
. y.
4052 0 0 1 1,PD 1 1,2 1,2,PD
1,2
4053 0 0 1 1 1 ";2y _ ~s".~ .y'
.
5051 0 0 1,2 ~~, ,,
~~:~ ~3,i- ~y2>3~. =- 33* _ ~
~~n_ _ >~, .
5052 0 0 1,2 1,2,3 . ~2b3~ >2,3~I~~.
~ ..1
~
x
5053 0 0 1 1 1 1,2 1,2,PD 1,2
6051 0 0 1 1 1
6052 0 0 1 1 1,PD 1~;2,
-, ,
',
6053 0 0 1 'l. = , ;~I31Qx,2,3 > ': ._~.~~:
, ~~~ lt~
~ ;
~
7051 0 1 1 1,3 ~ ~,; ,2,3~,. ,' 6
7052 0 1 1 1 v~~ 1~,
7053 0 1 3 I,3~1~ x ~I~ 1,2,3,ITIIr~~;~'~ ,= ~~~~~
~ ~~
u=mormai, 1=tall swollen, 1,=tail discoloration, ~=tatl necrotic, Yl)=partial
dose, UO=unable
to dose
+=Animals Dead
Conclusion
None of the formulations tested in the current study completely ameliorated
the
adverse effects of injection of Pseudomycin B. Complete amelioration has been
demonstrated with SBE-CD.
26
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Example 4 -- In vivo Toxicological Evaluation of an SBE-CD
Formulation with Varyin~ Concentrations of Pseudomycin B
This study evaluated potential toxicities, including any adverse effects at
the site
of injection, in an animal model of formulations of sulfobutylether-(3-
cyclodextrin at four
concentrations of pseudomycin B.
Materials and Method
The doses of pseudomycin B used for this rat study were 0, 1, 10, or 50
mg/kglday
for 14 days administered as an intravenous bolus dose. The SBE-CD formulation
was 2
wt-% and the dose volume used was 5 mLJkg. Toxicological parameters monitored
by
standard methods included body weights, clinical observations, mortality,
hematology,
clinical chemistry, limited pathology (liver, kidney, heart, and injection
site), and bone
marrow micronucleus (as a preliminary assessment of genetic toxicity).
Results
Within 2 days of starting the live phase, the tails of the high-dose animals
were
swollen and discolored to the point that further dosing became impossible
(Table 5). The
decision was made to pull these animals off study and to reevaluate the method
and
formulation used for high-dose toxicity assessment. The 1 and 10 mg/kg/day
dose groups
went the full 2 weeks and showed no evidence of overt toxicity.
27
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Table 5 -- Results of Administering SBE-CD Formulations at Three
Concentrations of Pseudomycin B.
PARAMETERS 1 m da 10 m da 50 m da
Clinical Observations- - Group terminated
on
study day 3 due
to
excessive tail vein
irntation
Mortalit - - -
Bod Wei ht - - BW
Gross Patholo - - -
Organ Weights - - Ki weight
Li, Ki, and T Li wei ht
Ht
Hematolo - -
Clinical Chemist- -
Histopathology - - Slight vacuolization
(Li, Ki, Ht, of
and IS) renal tubules
IS Inflammation/Necrosis
BM Micronucleus- ~ -
= No findings
_ = Parameter not evaluated due to early termination
Li = liver, Ki = kidney, Ht = heart, and IS = injection site
At termination, the high-dose animals grossly demonstrated marked tail vein
irritation. This was associated with decreased body weight and a
histopathology finding of
injection site inflammation and venous necrosis. Liver and kidney weights were
also
increased in these animals. There were no such findings in the mid- and low-
dose
animals. The bone marrow micronucleus test was negative in the low- and mid-
dose
animals (bone marrow micronucleus was not evaluated in the high-dose group due
to the
early termination). The only other finding was the histopathologic observation
of slight
vacuolization of the renal tubule observed in the high-dose animals.
Conclusion
Although the study reported in Example 2 demonstrated that formulation with
SBE-CD ameliorated the adverse effects of injection of pseudomycin B, adverse
effects
were observed with an SBE-CD formulation in the present study. However, the
dose
amount and concentration of pseudomycin B are significantly higher in the
present study.
28
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
This study evaluated doses of pseudomycin B up to 50 mg/kg, which is twice the
dose
tested in the studies reported in Example 2, which was 25 mg/kg. Furthermore,
the
present study injected this doubled dose of pseudomycin B in a volume only one
half that
used in the studies reported in Example 2. Thus, the concentration of
pseudomycin B in
the formulation increased 4-fold. The concentration of SBE-CD remained
constant at 2
wt-% in these studies, so the concentration of pseudomycin B increased 4-fold
relative to
the concentration of SBE-CD.
This study indicates that amelioration of adverse effects of injection
requires at
least a certain minimum concentration of SBE-CD relative to the concentration
of
pseudomycin B. Although not limiting to the present invention, this
observation is
consistent with formation of a complex of SBE-CD and pseudomycin B.
Example 5 -- Increasing the Concentration of SBE-CD at a
High Dose of Pseudomycin B Ameliorates Adverse Effects
This study investigated toxicities of high dose pseudomycin B with an
increased
amount of SBE-CD and determined that higher concentrations of the cyclodextrin
ameliorated the adverse effects of high dose pseudomycin B seen in Example 4.
Materials and Methods
Animals were as described in Example 4. The doses of pseudomycin B used for
this study were 0, 25, or 50 mg/kg/day administered for 14 days as an
intravenous slow
injection using a computer controlled pump to minimize test article
extravasation into
surrounding tissue. The vehicle used for this study was 4 wt-% SBE-CD and the
dose
volume was 10 mLJkg. These increases in the concentration of SBE-CD and the
dose
volume this adjusted the ratio of pseudomycin B to SBE-CD back to what it was
in the
studies reported in Example 3. The control group received 4 wt-% SBE-CD alone.
Body
weights, clinical observations, mortality, hematology, clinical chemistry,
limited
pathology (liver, kidney, heart, and injection site), and bone marrow
micronucleus were
monitored by standard methods.
29
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Rec»ltc
The live phase of this study was completed with no evidence of overt toxicity,
as
is shown by the results reported in Table 6.
Table 6 -- Results of Administering 4 wt-% SBE-CD Formulation with High
Concentrations of Pseudomycin B.
PARAMETERS 25 m da 50
m
da
Clinical Observations - -
Mortalit - -
Bod Wei ht - -
Gross Patholo - -
Organ Weights -I~Ki weight (9%) ~fKi
weight
(14%)
Li, Ki, and Ht
Hematology - decreased
erythron
values
(slight
anemia
Clinical Chemistry - ~I~ALT & AST (1.5X)
.cholesterol
~-
tot protein/albumin
~.
IBUN ve minor
Histopathology Slight vacuolizationMinor
of hepatocellular
(Li, Ki, Ht, and IS) renal tubules degeneration
Slight
vacuolization
of
renal
tubules
LBM Micronucleus - -
= No findings
_ = Parameter not evaluated due to early termination
Li = liver, Ki = kidney, Ht = heart, and IS = injection site
Conclusion
Increasing the ratio of SBE-CD to pseudomycin B in the formulation completely
ameliorated the irritation seen with the 50 mg/kg/day dose group in the study
reported in
Example 4. This is consistent with a mechanism of the cyclodextrin-mediated
protection
involving formation of a complex including pseudomycin B and the cyclodextrin,
which
results in protection of the vascular endothelium.
Amelioration of the adverse effects at the site of injection allowed the first
observation of apparent target organ effects at the highest tested dose of
pseudomycin B.
The hematology, clinical chemistry, and histopathology data indicate target
organ effects
associated with pseudomycin B administration. The development of a slight
anemia
suggests effects on red blood cells or red blood cell producing elements of
the bone
marrow. The liver also emerged as a potential target organ with evidence of
liver enzyme
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
leakage and hepatocellular injury and death. Both of these effects were seen
only at the
highest dose of 50 mg/kg/day. Increased kidney weight and slight vacuolization
of the
renal tubules was seen at both 25 and 50 mg/kg/day and may indicate renal
effects of
pseudomycin B.
Example 6 -- Evaluation of the Metabolism of Pseudomycin B
Following Intravenous Infection of a SBE-CD Formulation
This study evaluated metabolism of a formulation of pseudomycin B with
sulfobutylether-(3-cyclodextrin following intravenous injection in rats.
Materials and Method
The doses of pseudomycin B used for this rat study were 5 and 25 mg/kg
administered once as an intravenous bolus dose. The SBE-CD formulation was 2
wt-%
and the dose volume used was 5 mL/kg. Parameters monitored by standard methods
included area under the curve for the duration of detectable levels (AUC),
half-life, and
Cmax.
Results
The results of this study are reported in Table 7.
Table 7 -- Metabolism Studies of Formulations of Pseudomycin B With SBE-CD.
Dose of AUCo Half-lifeCmax
PSB Formulation (n hr/mL (hours) ( mL)
(m )
5 NaOAc buffer 48,799 4.2 17.0
5 2 wt-% SBE-CD 41,156 4.0 20.6
2 wt-% SBE-CD 60,873 4.0 85.9
Conclusions
The plasma concentrations of pseudomycin B were similar after a 5 mg/kg dose
in
25 either sodium acetate buffer or in a formulation with 2 wt-% SBE-CD. The
presence of 2
31
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
wt-% SBE-CD did not affect the plasma pharmacokinetic profile of pseudomycin B
in
comparison with acetate buffer. The formulation did not change any of the
measured
pharmacokinetic parameters. In the 2 wt-% SBE-CD formulation, increasing the
dose of
pseudomycin B from 5 to 25 mg/kg resulted in a near proportional increase in
C~X and
AUC. The plasma half life of pseudomycin B was independent of dose examined.
Measurable plasma concentrations of pseudomycin B were present for 24 hours
after the
administration of dose.
Example 7 -- In vivo Toxicological Evaluation of an HP-CD and a
Gamma-Cyclodextrin Formulation of Pseudomycin B
This study further evaluated the ability of the hydroxypropyl-(3-cyclodextrin
and
gamma-cyclodextrin formulations to provide protection from adverse effects
intravenous
injection of pseudomycin B.
Materials and Methods
Animals were male rats as described in Example 4 which were divided into four
groups of five. The doses of pseudomycin B used for this study were 0 and 50
mg/kg/day
for 14 days administered as an intravenous bolus in a dose volume of 10 mL/kg
at a rate
of 66 mL/hr. Hydroxypropyl-(3-cyclodextrin was obtained from Cyclodextrin
Technology
Inc. Gamma-cyclodextrin was obtained from blacker. The cyclodextrin
concentration
used in the vehicle preparations was 4 wt-% in pH 4.5 acetate buffer (O.OSM).
Control
groups received either 4 wt-% gamma-CD or 4 wt-% hydroxypropyl-CD lacking
pseudomycin B.
Results
The results of this study are reported in Table 8.
32
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Table 8 -- Results of Administering Hydroxypropyl-(3-Cyclodextrin and Gamma-
Cyclodextrin Formulations at Two Concentrations of Pseudomycin B.
PARAMETERS H Gamma-CD
drox
ro
1-B-CD
Clinical Observations2 Dosing terminated on
animals study day
demonstrated
slight
tail
vein 2 due to excessive
irritation tail vein
at
the
end
of
the
2-
week irritation
dosin
eriod
Mortalit ~~,~~~k
t
~~
~
=-
M,"~~~~~
~..:=
:
..
Body Weight Slight DecIrease in BW parameters
decrease
in
BW
parameters
~
i.e., i.e., .LBW 14.8% at
BW d6
3.2%
at
d13
Gross Pathology~I'Kidney
wt.,
absolute
(8%)
relative
11
%
Hematology Changes
consistent
with
a
mild
anemia
Clinical Chemistry~ ~Chol (~30%) and .~Tri
I (~30%)
~ .
I ALT and AST ( 15-22%
NSS)
T BUN (16%)
. Serum albumin 7%
Histopathology Renal
cortical
vacuolization
in
(Li, Ki, Ht, control
and IS) (grade
slight)
and
treated
(grade
moderate)
animals
Perivasculitis
occurred
at
the
injection
site
of
control
(grade
minimal)
and
treated
(grade
slight-
moderate)
animals.
Inflammation
of
the
muscle
and
vascular
necrosis
were
found
onl
in
treated
rats.
-. Shading indicates no effect on the indicated parameter
Shading indicates the indicated parameter was not measured due to early
termination
The gamma-CD vehicle did not provide adequate protection from adverse effects
of pseudomycin B in this study. After only the first dose swelling and
discoloration of the
tail was noted. This became severe enough to prevent dosing by day 2.
The hydroxypropyl-(3-CD vehicle provided protection from adverse effects of
pseudomycin B. As a result, daily doses of the hydroxypropyl-(3-CD formulation
were
administered for the full 2-weeks of the study. Adverse effects on the
erythron
component, liver, kidney, and lipid metabolism are consistent with previous
observations
following 2 weeks of dosing at the 50 mg/kg level using a SBE-CD-based vehicle
(Example 5). There was some evidence of slight adverse effect at the site of
injection at
the gross and microscopic level in the present study. This was not reported in
the
previous SBE-CD vehicle study, suggesting that the SBE-CD, under the
conditions tested,
may provide a greater degree of protection than hydroxypropyl-(3-CD vehicle.
33
SUBSTITUTE SHEET (RULE 26)
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
Conclusion
In this study, gamma-CD did not provide protection from the adverse effects of
injection of pseudomycin B. Hydroxypropyl-(3-CD protected these animals from
the
adverse effects of injection of pseudomycin B.
The invention has been described with reference to various specific and
preferred
embodiments and techniques. However, it should be understood that many
variations and
modifications may be made while remaining within the spirit and scope of the
invention.
All publications and patent applications in this specification are indicative
of the
level of ordinary skill in the art to which this invention pertains. All
publications and
patent applications are herein incorporated by reference to the same extent as
if each
individual publication or patent application was specifically and individually
indicated by
reference.
34
CA 02371942 2001-10-11
WO 00/62793 PCT/US00/08725
SEQUENCE LISTING
<110> Eli Lilly and Company
<120> Pseudomycin Antifungal Compositions and Methods for
Their Use
<130> X-11463 Sequence Listing
<140>
<141>
<150> 60/129,435
<151> 1999-04-15
<160> 1
<170> PatentIn Ver. 2.1
<210> 1
<211> 10
<212> PRT
<213> Pseudomonas syringae
<220>
<223> Xaa at position 5 is homoserine.
<220>
<223> Xaa at position 6 is dehydroaminobutyric acid.
<220>
<223> Xaa at position 8 is dehydroalanine.
<400> 1
Thr Ala Thr Gln Xaa Xaa Ala Xaa Thr Arg
1 5 10
1
