U.S. patent application number 17/045283 was filed with the patent office on 2021-06-10 for synthesis of novel xylose free occidiofungin analogues and methods of using them.
The applicant listed for this patent is SANO CHEMICALS, THE TEXAS A&M UNIVERSITY SYSTEM. Invention is credited to MENGXIN GENG, RAVI ORUGUNTY, AKSHAYA RAVICHANDRAN, JAMES L. SMITH.
Application Number | 20210169846 17/045283 |
Document ID | / |
Family ID | 1000005428184 |
Filed Date | 2021-06-10 |
United States Patent
Application |
20210169846 |
Kind Code |
A1 |
SMITH; JAMES L. ; et
al. |
June 10, 2021 |
SYNTHESIS OF NOVEL XYLOSE FREE OCCIDIOFUNGIN ANALOGUES AND METHODS
OF USING THEM
Abstract
The subject invention pertains to methods for producing novel
occidiofungin analogues lacking xylose (OF-.DELTA.xyl analogue).
OF-.DELTA.xyl analogues can contain aldehyde, amine, triazole, or
hydrazones. The aldehyde, amine, triazole or hydrazone containing
analogues of OF-.DELTA.xyl can be further modified by substitution
with additional functional groups. Various OF-.DELTA.xyl analogues
described herein have reduced inflammation and toxicity and
superior pharmacological properties compared to the natural
occidiofungins. Accordingly, certain embodiments of the invention
provide methods of treating or preventing fungal infections by
administering to the subjects in need thereof the OF-.DELTA.xyl
analogues described herein.
Inventors: |
SMITH; JAMES L.; (BRYAN,
TX) ; GENG; MENGXIN; (BEIJING, CN) ;
RAVICHANDRAN; AKSHAYA; (PALM BEACH GARDENS, FL) ;
ORUGUNTY; RAVI; (ROUND ROCK, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TEXAS A&M UNIVERSITY SYSTEM
SANO CHEMICALS |
COLLEGE STATION
BRYAN |
TX
TX |
US
US |
|
|
Family ID: |
1000005428184 |
Appl. No.: |
17/045283 |
Filed: |
April 5, 2019 |
PCT Filed: |
April 5, 2019 |
PCT NO: |
PCT/US2019/025989 |
371 Date: |
October 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62653253 |
Apr 5, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/12 20130101;
A61P 43/00 20180101; A61K 31/395 20130101 |
International
Class: |
A61K 31/395 20060101
A61K031/395; A61K 38/12 20060101 A61K038/12; A61P 43/00 20060101
A61P043/00 |
Goverment Interests
[0002] This invention was made with government support under
1R41A1122441-01A1 awarded by the NIH-NIAID. The government has
certain rights in the invention.
Claims
1-26. (canceled)
27. An analogue of an occidiofungin or salt thereof, the
occidiofungin having Formula I: ##STR00006## wherein the analog or
salt thereof does not contain xylose moiety from Formula I
(OF-.DELTA.xyl analogue).
28. The OF-.DELTA.xyl analogue or salt thereof claim 27, having
Formula II: ##STR00007##
29. The OF-.DELTA.xyl analogue or salt thereof claim 27, having
Formula III: ##STR00008##
30. The OF-.DELTA.xyl analogue or salt thereof of claim 29, wherein
R.sub.4 or R.sub.5 of Formula III is independently H, OH, a
substituted or unsubstituted alkane, substituted or unsubstituted
alkene, substituted or unsubstituted alkyne, substituted or
unsubstituted aryl, substituted or unsubstituted heterocycle,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycle, substituted or unsubstituted ether,
substituted or unsubstituted thioether, substituted or
unsubstituted ketone or a halogen.
31. The OF-.DELTA.xyl analogue or salt thereof of claim 27, having
Formula IV: ##STR00009##
32. The OF-.DELTA.xyl analogue or salt thereof of claim 27, having
Formula V: ##STR00010##
33. The OF-.DELTA.xyl analogue or salt thereof of claim 32, wherein
R.sub.6 of Formula V is phenyl, 4-nitro phenyl, 2-nitrophenyl or
2,4-dinitrophenyl.
34. The OF-.DELTA.xyl analogue or salt thereof of claim 33, wherein
R.sub.6 is 2-aminophenyl, 4-aminophenyl or 2,4-diaminophenyl,
wherein one or more amino group(s) is/are optionally
substituted.
35. The OF-.DELTA.xyl analogue or salt thereof of claim 34, wherein
the one or more amine group(s) of 2-aminophenyl, 4-aminophenyl or
2,4-diaminophenyl is/are substituted with OH, a substituted or
unsubstituted alkane, substituted or unsubstituted alkene,
substituted or unsubstituted alkyne, substituted or unsubstituted
aryl, substituted or unsubstituted heterocycle, substituted or
unsubstituted heteroaryl, substituted or unsubstituted heterocycle,
substituted or unsubstituted ether, substituted or unsubstituted
thioether, substituted or unsubstituted ketone or a halogen.
36. A pharmaceutical composition comprising an OF-.DELTA.xyl
analogue or salt thereof of claim 27 and a pharmaceutically
acceptable vehicle.
37. A method of treating or preventing a fungal infection,
comprising administering to a subject in need thereof an effective
amount of an OF-.DELTA.xyl analogue or salt thereof of claim 27 or
a pharmaceutical composition thereof.
38. The method of claim 37, comprising administering the
OF-.DELTA.xyl analogue or salt thereof or the pharmaceutical
composition intravaginally, intramuscularly, subcutaneously,
intrathecally, intravenously or intraperitoneally.
39. The method of claim 37, wherein the fungal infection is caused
by a Candida spp., Trichophyton spp., Rhizopus spp., Mucor spp.,
Fusarium spp., Aspergillus spp. or Cryptococcus spp.
40. The method of claim 39, wherein the Candida spp. is C.
albicans, C. glabrata, C. krusei, C. krusei, C. parapsilosis or C.
tropicalis.
41. A method of producing an OF-.DELTA.xyl analogue of Formula II
or salt thereof, comprising an oxidative cleavage between the fifth
and sixth carbons of NAA of an occidiofungin having Formula I,
wherein the oxidative cleavage is designed to convert the fifth
carbon to a carbonyl of a resulting aldehyde.
42. The method of claim 41, wherein the occidiofungin of Formula I
is treated with metaperiodic acid (HIO.sub.4), sodium periodate
(NaIO.sub.4) or potassium periodate (KIO.sub.4) for about 20-40
minutes at about 60-80.degree. C.
43. A method of producing an OF-.DELTA.xyl analogue of Formula III
or salt thereof, comprising a first step of treating the
OF-.DELTA.xyl analogue of Formula II with a primary or secondary
amine in the presence of an alcohol to produce an imine containing
OF-.DELTA.xyl analogue and a second step of reducing the imine
containing OF-.DELTA.xyl analogue with a reducing agent to produce
the OF-.DELTA.xyl analogue of Formula III.
44. The method of claim 43, wherein the alcohol is methanol, the
reducing agent is NaBH.sub.4, and the primary or secondary amine is
undecylamine, dodecylamine or DL-dihydrosphingosine.
45. A method of producing an OF-.DELTA.xyl analogue of Formula IV
or salt thereof, comprising reacting an OF-.DELTA.xyl analogue of
Formula II with a substituted or unsubstituted triazole.
46. The method of claim 45, comprising a first step of treating the
OF-.DELTA.xyl analogue of Formula II with the substituted or
unsubstituted triazole in the presence of an alcohol to produce an
intermediate OF-.DELTA.xyl analogue and a second step of reducing
the intermediate OF-.DELTA.xyl analogue with a reducing agent to
produce the OF-.DELTA.xyl analogue of Formula IV.
47. The method of claim 46, wherein the alcohol is methanol and the
reducing agent is NaBH.sub.4.
48. A method of producing an OF-.DELTA.xyl analogue of Formula V or
salt thereof, comprising reacting an OF-.DELTA.xyl analogue of
Formula II with a hydrazine.
49. The method of claim 48, comprising a first step of treating the
OF-.DELTA.xyl analogue of Formula II with a hydrazine in the
presence of an alcohol to produce an intermediate OF-.DELTA.xyl
analogue and a second step of reducing the intermediate
OF-.DELTA.xyl analogue with a reducing agent to produce the
OF-.DELTA.xyl analogue of Formula V.
50. The method of claim 49, wherein the alcohol is methanol and the
reducing agent is NaBH.sub.4.
51. The method of claim 48, wherein the hydrazine is phenyl
hydrazine, 2-nitrophenyl hydrazine, 4-nitrophenyl hydrazine or
2,4-dinitrophenyl hydrazine.
52. The method of claim 51, further comprising reducing one or more
nitro-groups from 2-nitrophenyl hydrazine, 4-nitrophenyl hydrazine
or 2,4-dinitrophenyl hydrazine to amines.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/653,253, filed Apr. 5, 2018, the disclosure
of which is hereby incorporated by reference in its entirety,
including all figures, tables and amino acid or nucleic acid
sequences.
[0003] The Sequence Listing for this application is labeled
"Seq-List.txt" which was created on Apr. 5, 2019 and is 1 KB. The
entire content of the sequence listing is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0004] Fungal infections caused by pathogens that are resistant to
commonly used classes of antifungals are becoming increasingly
prevalent. A CDC report described the spread of multi-drug
resistant Candida auris causing systemic infections in hospitalized
patients. Furthermore, other species such as Candida glabrata and
Candida parapsilosis have been reported to have gained resistance
to routinely used azoles and echinocandins. An example of a fungal
infection that is rapidly developing resistance to currently
available treatments is vulvovaginal candidiasis (VVC). VVC affects
approximately 75% of women and 5-10% of women develop recurrent VVC
(RVVC). Approximately 90% of VVC is caused by C. albicans, while
the remaining 10% is caused by C. glabrata, Candida tropicalis,
Candida parapsilosis and Candida krusei. These non-albicans VVC
causing species are generally resistant to azole treatments, which
are the most common treatment option for VVC. No new therapeutic
developments occurred in decades for RVVC. There is no established
treatment method for VVC, which has led to the development of
several ineffective treatment methods. These generally include the
use of a rigorous dosing regimen of antifungals followed by a long
period of prophylactic dosing.
[0005] Candida species are an important cause of infection and
mortality in all hospitalized patients. Candidemia has a mortality
rate of 30%-50% in cancer patients and is a major complicating
factor in the treatment of cancer. Despite current antifungal
drugs, invasive fungal infections are still a major cause of
morbidity and mortality in transplant patients. Reports suggest
that candidal infection is the first and second most common
infection in lung and heart transplant recipients, respectively. In
heart transplant recipients, candidal disease has been attributed
to a mortality rate of 28%. A rise in candidemia caused by
non-albicans Candida spp. and an increase in azole resistance is
alarming; and supports the need for new antifungals. More
appropriate antifungal treatment options may reduce the cost of
treatment and mortality of patients.
[0006] Antifungals currently in clinical use primarily comprise of
the polyenes, echinocandins and azoles. The polyene antifungal
amphotericin B was introduced in the 1950s and was the only
antifungal available until the introduction of the azole class of
antifungals in the 1980s. These two groups primarily target
ergosterol production or bind to ergosterol, disrupting the fungal
membrane. The echinocandins, the third group, are synthetically
modified lipopeptides that originate from a natural cyclic peptide
produced by fungi. This group selectively inhibits
1,3-.beta.-glucan synthesis by functioning as a non-competitive
inhibitor of 1,3-.beta.-glucan synthase. Widespread resistance and
the ineffective spectrum of activity of these antifungals have been
reported. The prevalence of echinocandin and azole-resistant fungal
pathogens and the limited spectrum of activity of those compounds
is one major issue contributing to the need for a new class of
antifungals. Additionally, current antifungal treatments lead to
abnormal liver and kidney function tests and have limitations with
respect to their spectrum of activity and toxicities. Presumably,
the identification of a novel class of antifungals with a broad
spectrum of activity and a unique mechanism of action would
mitigate the loss of life associated with the use of the current
classes of antifungals. These limitations and toxicity problems
have created an urgent need to identify antifungal compounds that
have new fungal cell targets.
[0007] Occidiofungins, isolated from Burkholderia contaminans MS14,
are a newly discovered class of antifungals. Occidiofungin is a
non-ribosomally synthesized glycolipopeptide produced by B.
contaminans MS14, a soil bacterium. It is a cyclic peptide with a
base mass of 1200 Da (FIG. 1). The bacterium produces a mixture of
structural analogues of the base compound (Occidiofungins A-D).
However, all analogues are composed of eight amino acids and a C18
fatty amino acid (hereinafter, NAA) containing a xylose sugar, and
a 2,4-diaminobutyric acid (DABA). The structural analogues differ
by the addition of oxygen to asparagine 1 (Asn1) forming a
.beta.-hydroxy asparagine 1 (BHN1) and by the addition of chlorine
to .beta.-hydroxy tyrosine 4 (BHY) forming 3-chloro .beta.-hydroxy
tyrosine 4 (chloro-BHY). Occidiofungins (A-D) show promise for the
development of novel therapeutics for treating fungal
infections.
[0008] Improvements in the production of the occidiofungin B analog
have been made. The method devised does not lead to a complex
mixture of occidiofungin.
[0009] Mammalian glycoproteins containing xylose are not reported.
Xylose is one of the main cross-reactive carbohydrate determinants
(CCDs) that lead to an allergic response. An allergic or stress
response was observed in mice following an intravenous
administration of occidiofungin (A-D). Therefore, producing xylose
free occidiofungin (OF-.DELTA.xyl) analogues and determining their
efficacy against fungal infections is desirable.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention provides methods for producing novel
occidiofungin analogues that lack xylose (OF-.DELTA.xyl analogues)
and methods of using OF-.DELTA.xyl analogues. In certain
embodiments, OF-.DELTA.xyl analogues are aldehyde containing
analogues. In certain embodiments, the aldehyde containing
OF-.DELTA.xyl analogues are further modified to produce amine
containing OF-.DELTA.xyl analogues, triazole containing
OF-.DELTA.xyl analogues or hydrazones containing OF-.DELTA.xyl
analogues. The aldehyde, amine, triazole or hydrazone containing
OF-.DELTA.xyl analogues can be further modified to produce
additional OF-.DELTA.xyl analogues. Various OF-.DELTA.xyl analogues
described herein induce reduced inflammation and toxicity and have
superior pharmacological properties compared to the natural
occidiofungins. Accordingly, certain embodiments of the invention
provide methods of controlling fungal infections by administering
to the subjects in need thereof the OF-.DELTA.xyl analogues
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Structure of occidiofungin. The location of the diol
is at carbons 5 and 6 (as numbered in the Figure).
[0012] FIGS. 2A-2D. A, B, C and D provide the structures of
aldehyde, amine, triazole and hydrazone containing OF-.DELTA.xyl
analogues, respectively.
[0013] FIGS. 3A-3E. A provides a scheme of oxidative cleavage for
the synthesis of aldehyde containing OF-.DELTA.xyl analogues, B
describes reductive amination for the synthesis of amine containing
OF-.DELTA.xyl analogues, C provides a scheme for the synthesis of
triazole containing OF-.DELTA.xyl analogues and D describes a
scheme for the synthesis of hydrazone containing OF-.DELTA.xyl
analogues, E describes a scheme for synthesis of three acyl lipid
analogs.
[0014] FIGS. 4A-4B. Generation and characterization of the aldehyde
analog of occidiofungin. (A) HPLC isolation of the aldehyde analog
of occidiofungin. The solvent used was 20% methanol in water. The
black line represents the absorbance unit (AU) at 220 nm. (B)
ESI-MS chromatograms of the aldehyde analog (868 Da) of
occidiofungin. The data shows that the oxidative cleavage went to
completion, given that there is no mass of starting material (1216
Da).
[0015] FIGS. 5A-5D. HPLC chromatograph of 100 .mu.g of wild-type
occidiofungin (A), aldehyde containing OF-.DELTA.xyl analogues
following the reaction with undecylamine (B), dodecylamine (C), and
DL-dihydrosphingosine D). The peak indicated by arrow is a desired
product. Grey lines represent the gradient of buffer B (water with
0.1% TFA), while black lines represent the absorbance unit (AU) at
220 nm.
[0016] FIGS. 6A-6E. ESI-MS of OF-.DELTA.xyl analogues. (A)
wild-type, (B) an aldehyde containing OF-.DELTA.xyl analogue, (C)
an aldehyde containing OF-.DELTA.xyl analogue following a reaction
with undecylamine, (D) an aldehyde containing OF-.DELTA.xyl
analogue following a reaction with dodecylamine, and (E) an
aldehyde containing OF-.DELTA.xyl analogue following a reaction
with DL-dihydrosphingosine.
[0017] FIG. 7. Efficacy of occidiofungin in treating murine
vulvovaginal candidiasis. The graph demonstrates CFUs per ml of
Candida albicans in the control group of mice compared to the
groups treated intravaginally with different concentrations of
occidiofungin in 0.3% noble agar. Error bars represent standard
deviation. Statistical analyses indicate a significant difference
between the control group and the treated groups (p<0.001) as
indicated by the asterisk.
[0018] FIGS. 8A-8B. TOCSY and NOESY NMR spectra of occidiofungin.
(A) TOCSY spin system correlations of the aldehyde occidiofungin
product. Fingerprint region (NH correlations), alpha to side chain
correlations and side chain correlations are shown. Abbreviations
are: diamino butyric acid 5 (DABAS), novel amino acid 2 (NAA2),
beta-hydroxy-asparagine 1 (BHN1), and beta-hydroxy-tyrosine 4
(BHY4). (B) NOESY spin system correlations. The expansion shows the
intra-residue NOE interaction of the aldehyde proton of NAA2 to the
amide proton of the NAA2.
[0019] FIGS. 9A-9D. Co-sedimentation assay demonstrating the
binding of occidiofungin analogs to actin. (A) Binding curve of
native occidiofungin to actin (Kd=1000 nM; the stoichiometry
[ligand:protein] is 25: 1. (B) Binding curve of the dodecylamine
analog to actin (Kd=4200 nM; the stoichiometry [ligand:protein] is
34: 1. (C) Binding curve of phalloidin to actin (Kd=8 nM; the
stoichiometry [ligand:protein] is 0.7:1. (D) Binding curve of the
dihydrosphingosine analog to actin (Kd=25 nM; the stoichiometry
[ligand:protein] is 1.8:1. The graph is plotted between the amount
of free compound obtained in the supernatant of the
co-sedimentation assay and the amount of bound compound obtained
from the actin pellet. Data for native occidiofungin and phalloidin
has been published previously (Ravichandran et al.).
[0020] FIGS. 10A-10B. Pyrene labeled actin polymerization and
depolymerization assay. The effect of phalloidin, native
occidiofungin, and the dihydrosphingosine analog on actin (A)
polymerization and (B) depolymerization assays. (grey .cndot.)
represents buffer baseline control; (.box-solid.) represents no
drug control; (.diamond-solid.) represents phalloidin treatment;
(black .cndot.) represents native occidiofungin treatment;
(.tangle-solidup.) represents dihydrosphingosine analog treatment.
The fluorescent data is measured in terms of arbitrary units
(AU).
BRIEF DESCRIPTION OF THE SEQUENCES
[0021] SEQ ID NO: 1: Sequence of cyclic occidiofungin antibiotic.
Asn1-[Novel Amino Acid 2 (NAA2)-Ser3-BHY4-Gly6-Asn7-Ser8].
DETAILED DISCLOSURE OF THE INVENTION
[0022] As used herein, the singular forms "a", "an" and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Further, to the extent that the terms
"including", "includes", "having", "has", "with", or variants
thereof are used in either the detailed description and/or the
claims, such terms are intended to be inclusive in a manner similar
to the term "comprising."
[0023] The phrases "consisting essentially of" or "consists
essentially of" indicate that the claim encompasses embodiments
containing the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s) of the
claim.
[0024] The term "about" means within an acceptable error range for
the particular value as determined by one of ordinary skill in the
art, which depend in part on how the value is measured or
determined, i.e., the limitations of the measurement system. Where
particular values are described in the application and claims,
unless otherwise stated the term "about" meaning within an
acceptable error range for the particular value should be assumed.
In the context of compositions containing amounts of ingredients
where the terms "about" or "approximately" are used, these
compositions contain the stated amount of the ingredient with a
variation (error range) of 0-10% around the value (X.+-.10%).
[0025] In the present disclosure, ranges are stated in shorthand,
so as to avoid having to set out at length and describe each and
every value within the range. Any appropriate value within the
range can be selected, where appropriate, as the upper value, lower
value, or the terminus of the range. For example, a range of
0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as
the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
and all intermediate ranges encompassed within 0.1-1.0, such as
0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two
significant digits within a range are envisioned, for example, a
range of 5-10 indicates all the values between 5.0 and 10.0 as well
as between 5.00 and 10.00 including the terminal values.
[0026] When ranges are used herein, such as for dose ranges,
combinations and subcombinations of ranges (e.g., subranges within
the disclosed range), specific embodiments therein are intended to
be explicitly included.
[0027] "Pharmaceutically acceptable" means approved or approvable
by a regulatory agency of the Federal or a state government or the
corresponding agency in countries other than the United States, or
that is listed in the U.S. Pharmacopoeia or other generally
recognized pharmacopoeia for use in animals, and more particularly,
in humans.
[0028] "Pharmaceutically acceptable salt" refers to a salt of an
occidiofungin analogue of the invention that is pharmaceutically
acceptable and that possesses the desired pharmacological activity
of the parent occidiofungin analogue. In particular, such salts are
non-toxic may be inorganic or organic acid addition salts and base
addition salts.
[0029] "Pharmaceutically acceptable vehicle" refers to a diluent,
adjuvant, excipient or carrier with which an occidiofungin analogue
of the invention is administered. A "pharmaceutically acceptable
vehicle" refers to a substance that is non-toxic, biologically
tolerable, and otherwise biologically suitable for administration
to a subject, such as an inert substance, added to a
pharmacological composition or otherwise used to facilitate
administration of an agent and that is compatible therewith.
Examples of vehicles include but are not limited to calcium
carbonate, calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils, and polyethylene
glycols.
[0030] "Subject" includes humans or non-human animals,
particularly, mammals, such as bovine, porcine, canine, rodent, or
feline animals.
[0031] "Treating" or "treatment" of any infection refers, in one
embodiment, to ameliorating the infection (i.e., arresting or
reducing the development of the disease or at least one of the
clinical symptoms thereof). In another embodiment "treating" or
"treatment" refers to ameliorating at least one physical parameter,
which may not be discernible by the subject. In yet another
embodiment, "treating" or "treatment" refers to modulating the
infection, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter), or both. In yet another embodiment, "treating" or
"treatment" refers to delaying the onset of the infection.
[0032] As used herein, the terms "reducing", "inhibiting",
"blocking", "preventing", "alleviating", or "relieving" when
referring to an occidiofungin analogue, mean that the occidiofungin
analogue brings down the occurrence, severity, size, volume or
associated symptoms of an infection by at least about 7.5%, 10%,
12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% compared to how the
infection would normally exist without application of the
OF-.DELTA.xyl analogue or a composition comprising the
OF-.DELTA.xyl analogue.
[0033] In treatment methods according to the invention, a
therapeutically effective amount of an OF-.DELTA.xyl analogues
according to the invention is administered to a subject suffering
from or diagnosed as having such an infection. A "therapeutically
effective amount" means an amount or dose sufficient to generally
bring about the desired therapeutic or prophylactic benefit in
patients in need of such treatment for the designated
infection.
[0034] Effective amounts or doses of the OF-.DELTA.xyl analogues of
the present invention may be ascertained by routine methods such as
modeling, dose escalation studies or clinical trials, and by taking
into consideration routine factors, e.g., the mode or route of
administration or drug delivery, the pharmacokinetics of the
occidiofungin analogue, the severity and course of the infection,
the subject's previous or ongoing therapy, the subject's health
status and response to drugs, and the judgment of the treating
physician. An example of a dose is in the range of from about 0.001
to about 200 mg of an occidiofungin analogue per kg of subject's
body weight per day, preferably about 0.05 to 100 mg/kg/day, or
about 1 to 35 mg/kg/day, even more preferably, about 1, 5, 10, or
20 mg/kg/day, in single or divided dosage units (e.g., BID, TID,
QID). For a 70-kg human, an illustrative range for a suitable
dosage amount is from about 0.05 to about 7 g/day, preferably,
about 0.07 to about 2.45 g/day, even more preferably, about 0.07,
0.35, 0.7, or 1.4 g/day.
[0035] Structure of natural analogues of occidiofungin is provided
by Formula I (also provided in FIG. 1):
##STR00001##
[0036] As seen in Formula I, natural analogues of occidiofungin are
composed of eight amino acids. One of these amino acids is NAA,
which contains a xylose sugar. NAA is 3
amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid. The carbon atoms
in NAA are numbered based on the carbonyl group of NAA as the first
carbon (FIG. 1). Also, the fifth and sixth carbon atoms of NAA each
have a hydroxyl group. The seventh carbon of NAA is connected to a
xylose via an ether linkage and a C.sub.11H.sub.23 moiety. Certain
structure and sequence information of occidiofungin is disclosed in
United States patent application publication number 2011/0136729,
which is herein incorporated by reference in its entirety. Certain
embodiments of the instant invention provide OF-.DELTA.xyl
analogues and methods of making OF-.DELTA.xyl analogues.
[0037] In one embodiment, an OF-.DELTA.xyl analogue comprises an
aldehyde group, wherein the fifth carbon of NAA is the carbonyl
group of the aldehyde, while the part of NAA from the sixth carbon
is removed from the parent occidiofungin. Such OF-.DELTA.xyl
analogue is referred to as aldehyde containing OF-.DELTA.xyl
analogue and it can be represented by Formula II (also provided in
FIG. 2A).
##STR00002##
[0038] In another embodiment, the carbonyl carbon of the aldehyde
containing OF-.DELTA.xyl analogue is aminated to produce an
analogue containing an amine group. Therefore, in such embodiments,
the fifth carbon of NAA is connected to an amine group while the
part of NAA beyond the sixth carbon is removed from the parent
occidiofungin. Such OF-.DELTA.xyl analogue is referred to as amine
containing OF-.DELTA.xyl analogue and can be represented by Formula
III (also provided in FIG. 2B).
##STR00003##
[0039] R.sub.4 and R.sub.5 in an amine containing OF-.DELTA.xyl
analogue can be H, OH, a substituted or unsubstituted alkane,
substituted or unsubstituted alkene, substituted or unsubstituted
alkyne, substituted or unsubstituted aryl, substituted or
unsubstituted heterocycle, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycle, substituted or
unsubstituted ether, substituted or unsubstituted thioether,
substituted or unsubstituted ketone or a halogen.
[0040] In certain embodiment, the aldehyde containing OF-.DELTA.xyl
analogues can be further modified to produce a triazole containing
OF-.DELTA.xyl analogue. In such embodiment, the fifth carbon of NAA
is connected to a triazole ring, while the part of NAA from the
sixth carbon and beyond is removed from the parent occidiofungin.
Such OF-.DELTA.xyl analogue is referred to as triazole containing
OF-.DELTA.xyl analogue and can be represented by Formula IV (also
provided in FIG. 2C).
##STR00004##
[0041] In certain embodiments, the aldehyde containing
OF-.DELTA.xyl analogues can be further modified to produce a
hydrazone containing OF-.DELTA.xyl analogue. In such embodiment,
the fifth carbon of NAA is connected to a hydrazine group, while
the part of NAA from the sixth carbon and beyond is removed from
the parent occidiofungin. Such OF-.DELTA.xyl analogue is referred
to as a hydrazone containing OF-.DELTA.xyl analogue and can be
represented by Formula V (also provided in FIG. 2D).
##STR00005##
[0042] Certain embodiments of the invention provide methods for
producing an aldehyde containing OF-.DELTA.xyl analogue. Such
methods comprise oxidative cleavage of NAA of a natural
occidiofungin between the fifth and sixth carbons while converting
the fifth carbon to a carbonyl of the resulting aldehyde.
Therefore, after an oxidative cleavage reaction, occidiofungin of
Formula I is converted into an OF-.DELTA.xyl analogue of Formula
II.
[0043] "Oxidative cleavage" as used herein comprises treating an
occidiofungin of Formula I with appropriate reagents under
appropriate conditions to cleave the carbon-carbon bond between the
fifth and the sixth carbons of NAA, and carbonyl group is formed on
the fifth carbon and optionally, on the sixth carbon.
[0044] In some embodiments, NAA is subjected to oxidative cleavage
between the fifth and sixth carbons with a metaperiodate, for
example, metaperiodic acid (HIO.sub.4), sodium periodate
(NaIO.sub.4), potassium periodate (KIO.sub.4), or a permanganate
salt, such as KMnO4. In certain embodiments, oxidative cleavage
between the fifth and sixth carbons with a periodate is performed
on carbohydrates in water. In specific embodiments, oxidative
cleavage between the fifth and sixth carbons is performed with a
periodate in mixed aqueous organic solvents, such as
acetonitrile/water, water/methanol and water/isopropanol. A skilled
artisan can determine an appropriate ratio of water and the organic
solvent.
[0045] Oxidative cleavage reaction with periodate converts the
fifth as well as the sixth carbons of NAA to carbonyl groups (FIG.
3A). Additional examples of periodates useful in such oxidative
cleavage are known to a skilled artisan and such embodiments are
within the purview of the invention.
[0046] In a specific embodiment, an occidiofungin of Formula I is
incubated with NaIO.sub.4 for about 20-40 minutes at about
60-80.degree. C. to produce an aldehyde containing OF-.DELTA.xyl
analogue of Formula II (FIG. 3A). The product can be further
purified, for example, via column chromatography, such as HPLC.
[0047] Additional reactions designed to cleave the NAA between the
fifth and sixth carbons while converting the fifth carbon to a
carbonyl group are known in the art and such embodiments are within
the purview of the invention.
[0048] Further embodiments of the invention also provide methods
for producing amine containing OF-.DELTA.xyl analogue (FIG. 2B). In
certain such embodiments, an amine containing OF-.DELTA.xyl
analogue is produced from an aldehyde containing OF-.DELTA.xyl
analogue. For example, an aldehyde containing OF-.DELTA.xyl
analogue is subject to a reductive amination with a primary or
secondary amine to produce an amine containing OF-.DELTA.xyl
analogue. This is a two-step reaction, with a first step involving
an imine formation with an alcohol, such as MeOH, followed by
reduction, for example, with NaBH.sub.4 (FIG. 3B). Amines suitable
for reductive amination include undecylamine, dodecylamine and
DL-dihydrosphingosine.
[0049] Additional reactions suitable for converting aldehyde
containing OF-.DELTA.xyl analogue to amine containing OF-.DELTA.xyl
analogue are well known to a person skilled in the art and such
embodiments are within the purview of the invention.
[0050] Further embodiments of the invention provide modifying amine
containing OF-.DELTA.xyl analogues, particularly, at R.sub.4 and
R.sub.5 positions of Formula III. For example, R.sub.4 and R.sub.5
in Formula III can be H, OH, a substituted or unsubstituted alkane,
substituted or unsubstituted alkene, substituted or unsubstituted
alkyne, substituted or unsubstituted aryl, substituted or
unsubstituted heterocycle, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycle, substituted or
unsubstituted ether, substituted or unsubstituted thioether,
substituted or unsubstituted ketone or a halogen. A skilled artisan
can design reactions for converting amine containing OF-.DELTA.xyl
analogues to corresponding analogues containing such substitutions
and such embodiments are within the purview of the invention.
[0051] Further embodiments of the invention also provide methods
for producing hydrazone containing OF-.DELTA.xyl analogue of
Formula V (FIG. 2D). An example of such reaction is provided in
FIG. 3C. In this reaction, an aldehyde containing OF-.DELTA.xyl
analogue is incubated with an alcohol, for example, methanol, at an
appropriate temperature, for example, about 20 to 35.degree. C.,
preferably, about 25.degree. C., for about 40 to 60 hours,
preferably, about 50 hours, even more preferably, about 48 hours.
This reaction produces an intermediate OF-.DELTA.xyl analogue,
which is reduced with an appropriate reducing agent, for example,
with NaBH.sub.4 (FIG. 3C). Triazole moiety illustrated in FIG. 3C
can be substituted or unsubstituted on one or more positions, for
example, by OH, a substituted or unsubstituted alkane, substituted
or unsubstituted alkene, substituted or unsubstituted alkyne,
substituted or unsubstituted aryl, substituted or unsubstituted
heterocycle, substituted or unsubstituted heteroaryl, substituted
or unsubstituted heterocycle, substituted or unsubstituted ether,
substituted or unsubstituted thioether, substituted or
unsubstituted ketone or a halogen.
[0052] Even further embodiments of the invention also provide
methods for producing hydrazone containing OF-.DELTA.xyl analogue
of Formula V (FIG. 2D). In certain embodiments, a hydrazone
containing OF-.DELTA.xyl analogue is produced from an aldehyde
containing OF-.DELTA.xyl analogue. An example of such reaction is
provided in FIG. 3D. In this reaction, an aldehyde containing
OF-.DELTA.xyl analogue is incubated with an alcohol, for example,
methanol, at an appropriate temperature, for example, about 20 to
35.degree. C., preferably, about 25.degree. C., for about 10 to 20
hours, preferably, about 15 hours, even more preferably, about 12
hours. This reaction produces an intermediate OF-.DELTA.xyl
analogue, which is reduced with an appropriate reducing agent, for
example, with NaBH.sub.4 (FIG. 3D). R.sub.4 or R.sub.5 in FIG. 3D
can be independently H, OH, a substituted or unsubstituted alkane,
substituted or unsubstituted alkene, substituted or unsubstituted
alkyne, substituted or unsubstituted aryl, substituted or
unsubstituted heterocycle, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycle, substituted or
unsubstituted ether, substituted or unsubstituted thioether,
substituted or unsubstituted ketone or a halogen.
[0053] In certain such embodiments, an aldehyde containing
OF-.DELTA.xyl analogue is treated with certain hydrazines, for
example, phenyl hydrazine, 2-nitrophenyl hydrazine, 4-nitrophenyl
hydrazine or 2,4-dinitrophenyl hydrazine. In certain such
embodiments, an appropriate hydrazine is dissolved in an aqueous
organic reaction mixture in the presence of a suitable Bronsted
acid or base or a suitable Lewis acid or base to produce hydrazine
containing OF-.DELTA.xyl analogues. Aqueous organic reaction
mixtures suitable in such embodiments include mixtures of
acetonitrile/water, water/methanol and water/isopropanol. A skilled
artisan can determine an appropriate ratio of water and the organic
solvent. Accordingly, under certain embodiments R.sub.6 group of
Formula V can be phenyl, 2-nitrophenyl, 4-nitrophenyl or
2,4-dinitrophenyl.
[0054] When present, the nitro-groups on the hydrazone containing
OF-.DELTA.xyl analogues, for example, analogs made from
2-nitrophenyl hydrazine, 4-nitrophenyl hydrazine or
2,4-dinitrophenyl hydrazine, can be further reduced to amines,
which in turn could be further modified. For example, the resulting
aromatic amines can be subjected to reductive amination with a
variety of aliphatic or aromatic aldehydes, such as acetaldehyde,
linear chain aldehydes, branched chain alkyl aldehydes and
benzaldehyde. Additional modifications include conjugation to OH, a
substituted or unsubstituted alkane, substituted or unsubstituted
alkene, substituted or unsubstituted alkyne, substituted or
unsubstituted aryl, substituted or unsubstituted heterocycle,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycle, substituted or unsubstituted ether,
substituted or unsubstituted thioether, substituted or
unsubstituted ketone or a halogen. A skilled artisan can design
reactions for converting amines from reduced hydrazone containing
OF-.DELTA.xyl analogues to corresponding analogues containing such
substitutions and such embodiments are within the purview of the
invention.
[0055] Salts of Occidiofungin Analogues of the Invention
[0056] In some embodiments the subject invention provides salts of
the OF-.DELTA.xyl analogues described herein. The salts can be a
salt with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid,
and phosphoric acid; an organic acid, such as trifluoroacetic acid
(TFA), formic acid, acetic acid, propionic acid, glycolic acid,
lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic
acid, maleic acid, and fumaric acid; or a salt with a base, such as
sodium hydroxide, ammonium hydroxide, potassium hydroxide, and
organic bases such as mono-, di-, trialkyl and aryl amines, and
substituted ethanolamines.
[0057] Further salts include: (1) acid addition salts, formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like; or
formed with organic acids such as acetic acid, propionic acid,
hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic
acid, lactic acid, malonic acid, succinic acid, malic acid, maleic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-di sulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary
butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic
acid, and the like; or (2) salts formed when an acidic proton
present in the parent occidiofungin analogue is replaced by a metal
ion, e.g., an alkali metal ion, an alkaline earth ion, or an
aluminum ion; or coordinates with an organic base such as
ethanolamine, diethanolamine, triethanolamine, N-methylglucamine
and the like. Salts further include, by way of example only,
sodium, potassium, calcium, magnesium, ammonium,
tetraalkylammonium, and the like; and when the occidiofungin
analogues contains a basic functionality, salts of non-toxic
organic or inorganic acids, such as hydrochloride, hydrobromide,
tartrate, mesylate, acetate, maleate, oxalate and the like.
[0058] Certain embodiments provide amorphous forms of salts of the
OF-.DELTA.xyl analogues disclosed herein. Such amorphous forms are
advantageous for oral, pulmonary, buccal, suppository delivery.
Routes of Administration and Dosage Forms
[0059] In certain embodiments, the OF-.DELTA.xyl analogues can be
administered intramuscularly, subcutaneously, intrathecally,
intravenously or intraperitoneally by infusion or injection.
Solutions of the occidiofungin analogues can be prepared in water,
optionally mixed with a nontoxic surfactant. Under ordinary
conditions of storage and use, these preparations can contain a
preservative to prevent the growth of microorganisms.
[0060] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the occidiofungin analogues that are
adapted for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. Preferably, the ultimate dosage form should be sterile,
fluid, and stable under the conditions of manufacture and storage.
The liquid carrier or vehicle can be a solvent or liquid dispersion
medium comprising, for example, water, ethanol, a polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycols,
and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained
by, for example, the formation of liposomes, by the maintenance of
the required particle size in the case of dispersions, or 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 is preferable to
include isotonic agents, for example, sugars, buffers, 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.
[0061] Sterile injectable solutions are prepared by incorporating
the OF-.DELTA.xyl analogues in the required amount in the
appropriate solvent as described herein with various of the other
ingredients enumerated herein, as required, preferably followed by
filter sterilization. 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 present in the previously sterile-filtered
solutions.
[0062] The compositions of the subject invention may also be
administered orally, in combination with a pharmaceutically
acceptable vehicle such as an inert diluent or an assimilable
edible carrier. They may be enclosed in hard or soft shell gelatin
capsules, may be compressed into tablets, or may be incorporated
directly with the food of the subject's diet.
[0063] For oral therapeutic administration, the OF-.DELTA.xyl
analogues can be combined with one or more excipients and used in
the form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 0.1% of a
occidiofungin analogue of the present invention. The percentage of
the occidiofungin analogues of the invention present in such
compositions and preparations may, of course, be varied and may
conveniently be between about 2% to about 60% of the weight of a
given unit dosage form. The amount of the OF-.DELTA.xyl analogues
in such therapeutically useful compositions is such that an
effective dosage level can be obtained.
[0064] The tablets, troches, pills, capsules, and the like may also
contain one or more of the following: binders such as gum
tragacanth, acacia, corn starch or gelatin; excipients such as
dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid, and the like; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose,
fructose, lactose, or aspartame, or a flavoring agent such as
peppermint, oil of wintergreen, or cherry flavoring may be
added.
[0065] When the unit dosage form is a capsule, it may contain, in
addition to materials of the above type, a liquid carrier, such as
a vegetable oil or a polyethylene glycol.
[0066] Various other materials may be present as coatings or for
otherwise modifying the physical form of the solid unit dosage
form. For instance, tablets, pills, or capsules may be coated with
gelatin, wax, shellac, or sugar, and the like. A syrup or elixir
may contain the an occidiofungin analogue, sucrose or fructose as a
sweetening agent, methyl and propylparabens as preservatives, a
dye, and flavoring such as cherry or orange flavor.
[0067] Of course, any material used in preparing any unit dosage
form should be pharmaceutically acceptable and substantially
non-toxic in the amounts employed.
[0068] In addition, the OF-.DELTA.xyl analogues may be incorporated
into sustained-release preparations and devices. For example, the
occidiofungin analogues may be incorporated into time release
capsules, time release tablets, time release pills, and time
release occidiofungin analogues or nanoparticles.
[0069] Pharmaceutical compositions for topical administration of
the OF-.DELTA.xyl analogues to the epidermis (mucosal or cutaneous
surfaces) can be formulated as ointments, creams, lotions, gels, or
as a transdermal patch. Such transdermal patches can contain
penetration enhancers such as linalool, carvacrol, thymol, citral,
menthol, t-anethole, and the like. Ointments and creams can, for
example, include an aqueous or oily base with the addition of
suitable thickening agents, gelling agents, colorants, and the
like. Lotions and creams can include an aqueous or oily base and
typically also contain one or more emulsifying agents, stabilizing
agents, dispersing agents, suspending agents, thickening agents,
coloring agents, and the like. Gels preferably include an aqueous
carrier base and include a gelling agent such as cross-linked
polyacrylic acid polymer, a derivatized polysaccharide (e.g.,
carboxymethyl cellulose), and the like.
[0070] Pharmaceutical compositions suitable for topical
administration in the mouth (e.g., buccal or sublingual
administration) include lozenges comprising the composition in a
flavored base, such as sucrose, acacia, or tragacanth; pastilles
comprising the composition in an inert base such as gelatin and
glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier. The pharmaceutical
compositions for topical administration in the mouth can include
penetration enhancing agents, if desired.
[0071] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina, and the
like. Other solid carriers include nontoxic polymeric nanoparticles
or microparticles. Useful liquid carriers include water, alcohols,
or glycols, or water/alcohol/glycol blends, in which the
occidiofungin analogues can be dissolved or dispersed at effective
levels, optionally with the aid of non-toxic surfactants. Adjuvants
such as fragrances and additional antimicrobial agents can be added
to optimize the properties for a given use. The resultant liquid
compositions can be applied from absorbent pads, used to impregnate
bandages and other dressings, or sprayed onto the affected area
using pump-type or aerosol sprayers.
[0072] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses, or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0073] Examples of useful dermatological compositions which can be
used to deliver the OF-.DELTA.xyl analogues to the skin are known
in the art; for example, see Jacquet et al. (U.S. Pat. No.
4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S.
Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508), all of
which are hereby incorporated by reference.
[0074] The concentration of the OF-.DELTA.xyl analogues of the
invention in such formulations can vary widely depending on the
nature of formulation and intended route of administration. For
example, the concentration of the occidiofungin analogues in a
liquid composition, such as a lotion, can preferably be from about
0.1-25% by weight, or, more preferably, from about 0.5-10% by
weight. The concentration in a semi-solid or solid composition such
as a gel or a powder can preferably be about 0.1-5% by weight, or,
more preferably, about 0.5-2.5% by weight.
[0075] Pharmaceutical compositions for spinal administration or
injection into amniotic fluid can be provided in unit dose form in
ampoules, pre-filled syringes, small volume infusion, or in
multi-dose containers, and can include an added preservative. The
compositions for parenteral administration can be suspensions,
solutions, or emulsions, and can contain excipients such as
suspending agents, stabilizing agents, and dispersing agents.
[0076] A pharmaceutical composition suitable for rectal
administration comprises OF-.DELTA.xyl analogues of the present
invention in combination with a solid or semisolid (e.g., cream or
paste) carrier or vehicle. For example, such rectal compositions
can be provided as unit dose suppositories. Suitable carriers or
vehicles include cocoa butter and other materials commonly used in
the art.
[0077] According to one embodiment, pharmaceutical compositions of
the present invention suitable for vaginal administration are
provided as pessaries, tampons, creams, gels, pastes, foams, or
sprays containing an occidiofungin analogue of the invention in
combination with carriers as are known in the art. Alternatively,
compositions suitable for vaginal administration can be delivered
in a liquid or solid dosage form.
[0078] Pharmaceutical compositions suitable for intra-nasal
administration are also encompassed by the present invention. Such
intra-nasal compositions comprise an occidiofungin analogue of the
invention in a vehicle and suitable administration device to
deliver a liquid spray, dispersible powder, or drops. Drops may be
formulated with an aqueous or non-aqueous base also comprising one
or more dispersing agents, solubilizing agents, or suspending
agents. Liquid sprays are conveniently delivered from a pressurized
pack, an insufflator, a nebulizer, or other convenient means of
delivering an aerosol comprising an occidiofungin analogue.
Pressurized packs comprise a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide, or other suitable gas as
is well known in the art. Aerosol dosages can be controlled by
providing a valve to deliver a metered amount of an occidiofungin
analogue.
[0079] The OF-.DELTA.xyl analogues may be combined with an inert
powdered carrier and inhaled by the subject or insufflated.
[0080] Pharmaceutical compositions for administration by inhalation
or insufflation can be provided in the form of a dry powder
composition, for example, a powder mix of an occidiofungin analogue
and a suitable powder base such as lactose or starch. Such powder
composition can be provided in unit dosage form, for example, in
capsules, cartridges, gelatin packs, or blister packs, from which
the powder can be administered with the aid of an inhalator or
insufflator.
[0081] The exact amount (effective dose) of the OF-.DELTA.xyl
analogues varies from subject to subject, depending on, for
example, the species, age, weight, and general or clinical
condition of the subject, the severity or mechanism of any
infection being treated, the particular agent or vehicle used, the
method and scheduling of administration, and the like. A
therapeutically effective dose can be determined empirically, by
conventional procedures known to those of skill in the art. See,
e.g., The Pharmacological Basis of Therapeutics, Goodman and
Gilman, eds., Macmillan Publishing Co., New York. For example, an
effective dose can be estimated initially either via in vivo assays
or in suitable animal models. The animal model may also be used to
determine the appropriate concentration ranges and routes of
administration. Such information can then be used to determine
useful doses and routes for administration in humans. Methods for
the extrapolation of effective dosages in mice and other animals to
humans are known to the art; for example, see U.S. Pat. No.
4,938,949, which is hereby incorporated by reference. A therapeutic
dose can also be selected by analogy to dosages for comparable
therapeutic agents.
[0082] The particular mode of administration and the dosage regimen
can be selected by the attending clinician, taking into account the
particulars of the case (e.g., the subject, the disease, the
disease state involved, and whether the treatment is prophylactic).
Treatment may involve daily or multi-daily doses of compound(s)
over a period of a few days to months, or even years.
[0083] In general, however, a suitable dose can be in the range of
from about 0.001 to about 100 mg/kg of body weight per day,
preferably from about 0.01 to about 100 mg/kg of body weight per
day, more preferably, from about 0.1 to about 50 mg/kg of body
weight per day, or even more preferred, in a range of from about 1
to about 10 mg/kg of body weight per day. For example, a suitable
dose may be about 1 mg/kg, 10 mg/kg, or 50 mg/kg of body weight per
day.
[0084] The OF-.DELTA.xyl analogues can be conveniently administered
in unit dosage form, containing for example, about 0.05 to about
10000 mg, about 0.5 to about 10000 mg, about 5 to about 1000 mg, or
about 50 to about 500 mg of active ingredient per unit dosage
form.
[0085] The OF-.DELTA.xyl analogues can be administered to achieve
peak plasma concentrations of, for example, from about 0.25 to
about 200 .mu.M, about 0.5 to about 75 .mu.M, about 1 to about 50
.mu.M, about 2 to about 30 .mu.M, or about 5 to about 25 .mu.M.
Exemplary desirable plasma concentrations include at least 0.25,
0.5, 1, 5, 10, 25, 50, 75, 100 or 200 .mu.M. For example, plasma
levels may be from about 1 to about 100 micromolar or from about 10
to about 25 micromolar. This may be achieved, for example, by the
intravenous injection of a 0.05 to 5% solution of an OF-.DELTA.xyl
analogue, optionally in saline, or orally administered as a bolus
containing about 1 to about 100 mg of the OF-.DELTA.xyl analogue.
Desirable blood levels may be maintained by continuous or
intermittent infusion.
[0086] The OF-.DELTA.xyl analogues can be included in the
compositions within a therapeutically useful and effective
concentration range, as determined by routine methods that are well
known in the medical and pharmaceutical arts. For example, a
typical composition can include one or more of the OF-.DELTA.xyl
analogues at a concentration in the range of at least about 1
mg/ml, preferably at least about 4 mg/ml, more preferably at least
5 mg/ml and most preferably at least 6 mg/ml.
[0087] The OF-.DELTA.xyl analogues may conveniently be presented in
a single dose or as divided doses administered at appropriate
intervals, for example, as one dose per day or as two, three, four
or more sub-doses per day. The sub-dose itself may be further
divided, e.g., into a number of discrete loosely spaced
administrations; such as multiple inhalations from an
insufflator.
[0088] Optionally, the pharmaceutical compositions of the present
invention can include one or more other therapeutic agents, e.g.,
as a combination therapy. The additional therapeutic agent(s) will
be included in the compositions within a therapeutically useful and
effective concentration range, as determined by routine methods
that are well known in the medical and pharmaceutical arts. The
concentration of any particular additional therapeutic agent may be
in the same range as is typical for use of that agent as a
monotherapy, or the concentration may be lower than a typical
monotherapy concentration if there is a synergy when combined with
an occidiofungin analogue of the present invention.
Methods of Treatment
[0089] As discussed above, the OF-.DELTA.xyl analogues disclosed
herein exhibit anti-fungal activity, particularly, against Candida
spp. Accordingly, certain embodiments of the invention provide
methods of administering an OF-.DELTA.xyl analogue to a subject in
need thereof to treat or prevent an infection, particularly, a
fungal infection, more particularly, a yeast infection, and even
more particularly, a Candida infection. In specific embodiments,
the invention provides methods of treating or preventing an
infection caused by a fungus selected from Trichophyton
mentagrophytes, Trichophyton rubrum, Rhizopus microspores, Mucor
circinelloides, Mucor fragilis, Fusarium solani, Fusarium
oxysporum, Aspergillus flavus, Aspergillus fumigatus, Candida
albicans, Candida glabrata, Candida krusei, Candida krusei, Candida
parapsilosis, Candida tropicalis or Cryptococcus neoformans.
Particularly, methods are provided for treating or preventing an
infection with any strain listed in Table 8.
[0090] Methods for treating or preventing an infection can be
performed in any subject, such as a mammal, including humans. Such
methods comprise administering to a subject in need of such
prevention or treatment of an infection an effective amount of an
OF-.DELTA.xyl analogue the subject invention. An OF-.DELTA.xyl
analogue can be administered in the form of a pharmaceutical
composition of an OF-.DELTA.xyl analogue.
[0091] Preferably, an occidiofungin analogue is administered
parenterally or enterally. Even more preferably, an occidiofungin
analogue is administered intravaginally, intraperitoneally,
subcutaneously or intravenously. The dosage of the effective amount
of an occidiofungin analogue can vary depending upon the age and
condition of each subject to be treated. However, suitable unit
dosages typically range from about 0.01 to about 100 mg. For
example, a unit dose can be in the range of about 0.2 mg to about
50 mg. Such a unit dose can be administered more than once a day,
e.g., two or three times a day.
Materials and Methods
[0092] Occidiofungin was purified as previously described by Lu et
al. Purified rabbit skeletal muscle filamentous actin (AKF99) was
purchased from Cytoskeleton Inc. (Denver, Colo.). Sodium periodate
(311448-5G), sodium borohydride (452882), undecylamine
(94200-10ML), dodecylamine (325163-5ML), and DL-dihydrosphingosine
(D6783-10MG) were purchased from Sigma (St. Louis, Mo.).
[0093] Synthesis of Occidiofungin Analogs
[0094] Occidiofungin was dissolved in 50% acetonitrile (ACN) with
0.1% trifluoroacetic acid (TFA) at a concentration of 1 mg/mL.
Sodium periodate was dissolved in ddH.sub.2O at a concentration of
1 mg/mL. Equal volumes of the two solutions were mixed thoroughly
in a 10 mL centrifuge tube and was incubated at 70.degree. C. for
30 min. One milliliter of the reaction mixture was loaded on a
BioRad Duoflow chromatography system with an analytical C18 column
(Agilent.RTM. ZORBAX, (Agilent Technologies, Santa Clara, Calif.
ODS, C18, 5 .mu.m, 4.6.times.250 mm). The reaction mixture was
separated with an isocratic flow of 20% MeOH in water and the
resulting aldehyde analog of occidiofungin eluted at approximately
9 minutes. The desired product was freeze-dried and weighed on an
analytical balance (Adventurer.TM. Pro AV114C, Ohaus Corporation,
USA).
[0095] A two-step procedure of reductive amination was used to
introduce a primary or secondary amine onto the aldehyde analog.
The first step of reaction involves the formation of an
intermediate carbinol amine, which is then dehydrated and
protonated to form an iminium ion. Subsequent reduction of this
iminium ion with sodium borohydride produces an alkylated amine
product. The aldehyde analog of occidiofungin was dissolved in DMSO
at a concentration of 10 mg/mL. Amines used were undecylamine,
dodecylamine, and DL-dihydrosphingosine. Aldehyde analog (10 .mu.L
of DMSO stock solution; 100 .mu.g) was mixed with 10-fold excess of
an amino lipid (molar ratio) in 400 .mu.L methanol. The sample was
then incubated at room temperature for at least 16 hours. Solid
sodium borohydride (6 mg) was weighted on an analytical balance
(Adventurer.TM. Pro AV114C, Ohaus Corporation, USA) and was
directly transferred into the reaction mixture, mixed well, and was
incubated at room temperature for 5 minutes. The reaction mixture
was diluted to 1 mL with 50% ACN 0.1% TFA and was separated by HPLC
using a 30-minute gradient from 90% to 20% water/ACN on the
analytical C18 column. Both HPLC solvents contained 0.1% TFA.
Products that eluted between 50-30% water were collected and
analyzed by electrospray ionization mass spectrometry (ESI-MS) on a
ThermoFisher DecaXP ion trap mass spectrometer. The yield of each
sample was quantified by comparing the peak area of each analog to
the peak area of a 100 .mu.g standard of occidiofungin.
[0096] NMR Spectroscopy
[0097] NMR analysis of aldehyde occidiofungin analog was performed
on a 5 mM sample dissolved in dimethyl sulfoxide (DMSO)-d6. The NMR
data were collected on an Avance III HD-600 with a TCI Cryoprobe
and an Avance III HD-850 with a TCI Cryoprobe. The .sup.1H
resonances were assigned according to standard methods using COSY,
TOCSY, NOESY, and .sup.13C-HSQC experiments. NMR experiments were
collected at 25.degree. C. .sup.1H chemical shifts were referenced
to DMSO peak at 2.5 ppm. The TOCSY experiment was acquired with a
60 ms mixing time using the Bruker DIPSI-2 spinlock sequence. The
NOESY experiment was acquired with a 400 ms mixing time. Phase
sensitive indirect detection for NOESY, TOCSY, and COSY experiments
was achieved using the standard Bruker pulse sequences. Peaks were
assigned using NMRView.
[0098] Minimum Inhibitory Concentration Assays
[0099] The minimal inhibitory concentration (MIC) is the lowest
concentration of compound that inhibits the visible growth of the
yeast after 24 hours of incubation. MIC assays were performed in
duplicate as previously described following a modified version of
the CLSI M27-A3 methods.
[0100] Actin Co-Sedimentation Assay
[0101] Actin binding experiments were performed as previously
described for native occidiofungin by Ravichandran et al. An
Agilent 1200 front end chromatography system and a TSQ Quantum.TM.
Access Triple Quadrupole Mass Spectrometer was used to analyze
phalloidin, native occidiofungin, dodecylamine analog, and the
DL-dihydrosphingosine analog binding to F-actin. Following a 10
.mu.L injection, samples containing dodecylamine analog of
occidiofungin were separated using a 15-minute water/ACN
(containing 0.2% formic acid) gradient starting from 95% to 40%
water on a C18 column (SinoChrom ODS-BP 5 .mu.m, 2.1 mm.times.50
mm). Samples containing DL-dihydrosphingosine analog were separated
on the same column through a modified water/ACN gradient starting
from 95% to 56% water over 5 minutes, from 56% to 52% water over 2
minutes, followed by a linear gradient from 52% to 48% water over 1
minute. The mass spectrometer was operated in positive mode and
operated using a protocol optimized for each compound. Briefly, the
center mass for dodecylamine analog was 1037.66 Da and the scan
width was 0.3 Da. The center mass for dihydrosphingosine analog was
1153.75 Da and the scan width was 1.0 Da. Area of each compound was
measured through manual integration using Xcalibur.TM. Software
(Thermo Fisher Scientific). The standard curves were generated for
each compound following the extraction protocol described above.
Native occidiofungin served as the internal standard for all
analogs. The R.sup.2 values for each standard curve exceeded
0.98.
[0102] Actin Polymerization and Depolymerization Assay
[0103] Actin polymerization and depolymerization assays were
performed using the Actin polymerization biochem kit (#BK003;
Cytoskeleton, Inc. Denver, Colo.) following the manufacturer's
instructions with some modifications. Briefly, for the
polymerization assay, G-actin stock solution was made at 0.2 mg/mL
instead of 0.4 mg/mL. For the depolymerization assay, F-actin stock
solution was diluted 9-fold instead of 5-fold. Test compounds
include phalloidin, dihydrosphingosine analog, and native
occidiofungin, at a final concentration of 5 .mu.M. The experiments
were done in duplicate.
[0104] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0105] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1--Synthesis of Semisynthetic Analogues of
Occidiofungin
[0106] Occidiofungin was produced and purified as described by Lu
et al. and Emrick et al. Chemicals were purchased from Sigma unless
otherwise indicated. Wild-type MS14 strain and xylose transferase
mutant strain used were obtained from glycerol stocks and cultured
on Yeast Peptone Dextrose (YPD) agar plate or broth at 35.degree.
C. Occidiofungin was produced from these cultures and used to
produce OF-.DELTA.xyl analogues as described herein.
[0107] Cleavage of diols on carbons 5 and 6 of the novel amino acid
yields an aldehyde containing OF-.DELTA.xyl analogue. Sodium
periodate breaks apart vicinal diols to form an aldehyde containing
analogue of OF-.DELTA.xyl (FIGS. 3A-3B). Occidiofungin was
dissolved in 50% ACN with 0.1% TFA at a concentration of 1 mg/mL.
Sodium periodate was dissolved in ddH.sub.2O at a concentration of
1 mg/mL. Equal volume of the two solutions were mixed in a
centrifuge tube and incubated at various temperatures from
30-70.degree. C. for 30 min. The reaction mixture was loaded on a
BioRad Duoflow.TM. chromatography system with an analytical column
(Agilent.RTM. ZORBAX, Agilent Technologies, Santa Clara, Calif.
ODS, C18, 5 .mu.m, 4.6.times.250 mm). The reaction mixture was
chromatographed with an isocratic flow of 20% MeOH in ddH.sub.2O.
The aldehyde containing analogue of OF-.DELTA.xyl elutes at
approximately 9 minutes (FIG. 4). The desired product is dried and
weighed on analytical balance (Adventurer.TM. Pro AV114C, Ohaus
Corporation, USA). The efficiency of the reaction and recovery of
the desired aldehyde-occidiofungin product was greater than
90%.
[0108] Reductive amination restores the aliphatic structural
element of occidiofungin through a condensation reaction with the
free amine. A two-steps procedure of reductive amination of
aldehyde containing OF-.DELTA.xyl analogue (FIG. 2A) was used to
introduce a primary or secondary amine at aldehyde. The first step
of reaction involves the formation of an intermediate carbinol
amine, which then undergoes dehydratation and protonation to form
an iminium ion. Subsequent reduction of this iminium ion with
sodium borohydride produces an alkylated secondary or tertiary
amine product depending on the nature of the starting material.
[0109] Chemicals used produce certain OF-.DELTA.xyl analogues
include undecylamine, dodecylamine, and DL-dihydrosphingosine. For
example, aldehyde containing OF-.DELTA.xyl analogue is dissolved in
DMSO at a concentration of 10 mg/mL and mixed with 10-fold excess
of undecylamine (molar ratio) in pure methanol and was incubated at
room temperature overnight. Solid sodium borohydride (6 mg) was
added to the overnight reaction and incubated at room temperature
for 5 minutes. The resulting product in the reaction mixtures was
chromatographically purified using a linear water/ACN gradient
starting from 90% to 20% water over 30-minute period. Both solvents
used in the mobile phase contained 0.1% trifluoroacetic acid (TFA).
The products eluted between 50-30% water with 0.1% TFA (FIG. 5).
The reaction efficiencies were determined by comparing the peak
area of each sample to the peak area of a 100 .mu.g standard of
occidiofungin (Table 1). The efficiency of the reactions and
isolation of the desired products were greater than 50% for all
reactions.
[0110] The molecular weight of the purified product was determined
by electrospray ionization mass spectrometry (ESI-MS) on a
ThermoFisher DecaXP ion trap mass spectrometer (Table 1). Briefly,
around 1 .mu.g of dried sample was resuspended in 100 .mu.l of 50%
ACN/water (vol/vol) and analyzed by direct infusion in positive
mode. MS data were recorded in a 0.5 min time frame (FIG. 6).
Occidiofungin wild-type was used as a comparison and mass spectral
control. Nuclear magnetic resonance studies were done to verify the
structure of the OF-.DELTA.xyl analogue.
TABLE-US-00001 TABLE 1 Summary of reactions. The starting material,
type of reaction, and yield of reaction are shown. The products are
confirmed by ESI-MS, all measured mass are the same with expected.
Yield of oxidation reaction was determined based on dry weight of
both starting material and product, while the yields of reductive
amination reactions were based on the peak volume of both starting
material and product on HPLC. Molecular Weight Starting Type Yield
(Da) material of reaction Amine (%) Expected Measured wild-type
oxidation N.A. 90 868 868 aldehyde reductive undecylamine 70 1024
1024 variant amination aldehyde reductive dodecylamine 84 1038 1038
variant amination aldehyde reductive DL-dihydro- 51 1154 1154
variant amination sphingosine
[0111] The removal of the aliphatic chain resulted in a highly
polar compound (FIGS. 1-2), which was isolated by HPLC under
isocratic conditions. Oxidative cleavage of the vicinal diols
proceeded efficiently, and occidiofungin was completely converted
to the aldehyde product (FIG. 2 and FIG. 3A). This was confirmed by
mass spectrometry of the reaction mixture (FIG. 4B). The isolated
aldehyde has a mass of 868 Daltons (Da). Nuclear magnetic resonance
(NMR) analyses of the isolated product further confirmed the
isolation of the cyclic peptide with the aldehyde on carbon 5 (C5)
of the NAA2 residue. The individual amino acid spin systems in the
2D TOCSY spectra are shown in (FIG. 8A) and the chemical shifts for
the aldehyde product are provided in Table 2. The through-space
proton interaction between the aldehyde proton and the amide proton
of the novel amino acid was observed in the 2D NOESY spectra (FIG.
8B), supporting the proton chemical shift assignments of the
aldehyde product. The aldehyde occidiofungin product was
subsequently used in reductive amination reactions affording the
synthesis of new analogs of occidiofungin.
[0112] Reductive amination reactions using three commercially
available long chain alkyl amines and the aldehyde occidiofungin
products were performed (FIG. 3B). The oxidative cleavage of the
diol in the NAA2 resulted in a loss of thirteen carbons in the
aliphatic chain. Since the xylose itself is not required for the
antifungal activity, undecylamine and dodecylamine were used to
restore the length of the aliphatic chain. A secondary amine is
incorporated at the carbon six (C6) position and an aliphatic chain
of eleven and twelve carbons were introduced for the undecylamine
and dodecylamine products, respectively (FIG. 3E). The alkyl amine
dihydrosphingosine was chosen because a hydroxyl group would be
reintroduced into the side chain near the normally occurring
positions found within the native product (FIG. 3E). The
unoptimized reactions were very efficient. The yield of the
semisynthetic undecylamine, dodecylamine, and dihydrosphingosine
analogs were 70, 80, and 50%, respectively (Table 2, FIGS. 5-6).
The final undecylamine, dodecylamine, and dihydrosphingosine
occidiofungin products had the expected masses of 1023.7, 1037.7,
and 1153.9 Da, respectively (Table 2, FIG. 5-6). Additional analogs
can be rapidly synthesized by this approach and the structural
diversity of analogs made can be expanded by the synthesis of novel
amino lipid compounds that are not readily available.
TABLE-US-00002 TABLE 2 NMR chemical shifts for aldehyde analog of
occidiofungin. Proton chemical shift values were from a TOCSY and
NOESY experiments. Amino Acid H.sup.N H.sup..alpha. H.sup..beta.
Other protons BHN1 8.11 4.57 4.09 .beta.-OH: 5.68, .gamma.-NH2:
7.20, 6.70 NAA2 7.76 C2: CH2- 7.58 2.08&1.92, C3: CH- 4.39, C4:
CH2-2.96, C5: CH-8.71 Ser3 8.12 4.27 3.56 .beta.-OH: 4.99 BHY4 8.02
4.45 4.04 .beta.-OH: 5.71, OH-9.28, C2&C6: CH-7.16, C3&C5:
CH-6.70 DABA5 7.61 4.23 2.02 .gamma.-H: 2.91, NH2: 7.71 .gamma.-H:
2.87, NH2: 7.66 Gly6 8.07 3.82, 3.64 Asn7 8.16 4.59 2.58, 2.47
.gamma.-NH2: 7.44, 6.97 Ser8 8.01 4.20 3.61 .beta.-OH: 5.08
Example 2--Minimum Inhibitory Concentration (Mic) of the
OF-.DELTA.xyl Analogues
[0113] Minimum inhibitory concentration (MIC) susceptibility
testing was performed following a modified version, as previously
reported, of the CLSI M27-A3 methods for the susceptibility testing
of yeasts. Incubation temperature was 35.degree. C. and the
inoculum size was 0.5-2.5.times.10.sup.3 colony-forming units
(CFU)/mL for yeasts. MICs were performed to determine the
bioactivity of each new OF-.DELTA.xyl analogue (Table 3). The
minimal inhibitory concentration (MIC) is the lowest concentration
of compound that inhibits the visible growth of the yeast after 24
hours of incubation and the assays were performed in duplicate. The
aldehyde containing OF-.DELTA.xyl analogues with the addition of an
undecylamine, dodecylamine, or DL-dihydrosphingosine did not
demonstrate any inhibitory activity against C. albicans at the
concentration tested. However, the addition of
DL-dihydrosphingosine to the aldehyde containing OF-.DELTA.xyl
analogue exhibited a low micromolar inhibitory activity against
Saccharomyces cerevisiae and Candida glabrata.
[0114] The aldehyde, undecylamine, and dodecylamine analogs were
inactive at concentrations 64-fold higher than the inhibitory
concentration of native occidiofungin against the Candida species
tested and S. cerevisiae strain tested (Table 3). The undecylamine
and dodecylamine analogs have a similar aliphatic carbon length as
native occidiofungin. Further, these analogs have similar polarity
as native occidiofungin, as was observed by the HPLC retention
times (FIGS. 5-6). The major structural differences to native
compound are the conversion of a (C5)-(C6) to (C5)-(N6) bond in the
side chain of NAA2, the removal of the vicinal diol group, and the
loss of a xylose sugar. Dihydrosphingosine analog was synthesized
to test whether the introduction of a hydroxyl group near (C6)
position could restore activity. Dihydrosphingosine analog has one
branched alcohol on (C7) and a hydroxyl on (C8) positions normally
found within native occidiofungin. Further, the aliphatic chain of
dihydrosphingosine analog is five carbons longer than the native
compound. The dihydrosphingosine restores some antifungal activity
as observed by a low micromolar inhibitory activity against S.
cerevisiae, C. albicans, and Candida glabrata; the MICs were 2, 16,
and 8 .mu.g/mL, respectively. The loss of activity, or the
reduction of activity, of the semisynthetic analogs of
occidiofungin could be attributed to a reduction in their ability
to penetrate the plasma membrane to reach cellular target (binding
to cell envelope or inability to cross plasma membrane) or a loss
in affinity to actin (the cellular target of occidiofungin).
TABLE-US-00003 TABLE 3 MIC (.mu.g/ml) of occidiofungin analogues
against Candida glabrata ATCC2001, Candida albicans ATCC 3147 and
S. cerevisiae DGY6 haploid BY4741. Sacchaomyces cerevisiae Candida
occidiofungin DGY6 haploid glabrata Candida albicans analogues
BY4741 ATCC 2001 ATCC 3147 wild-type 0.0625 0.5 0.5 aldehyde
containing >4 -- >8 OF-.DELTA.xyl analogue Undecylamine >4
-- >8 containing OF-.DELTA.xyl analogue Dodecylamine >4 --
>8 containing OF-.DELTA.xyl analogue DL-dihydrosphingosine 2 8
>8 containing OF-.DELTA.xyl analogue
Example 3--Additional Examples OF-.DELTA.xyl Analogues
[0115] The aldehyde containing OF-.DELTA.xyl analogue can be used
to create new analogues of occidiofungin besides the amine
containing OF-.DELTA.xyl analogues described above. Synthesis of
triazoles can be readily accomplished by addition of various aryl
azide in the presence of Cu(ACAC) as a catalyst. This allows for
the introduction of various aryl groups and conversion of the
aldehyde into a triazole.
[0116] Synthesis of hydrazones: The aldehyde occidiofungin (1) can
be treated with a variety of phenyl hydrazines such as i)
2-nitrophenyl hydrazine, 4-nitrophenyl hydrazine or
2,4-dinitrophenyl hydrazine (2,4 DNPH) to produce novel hydrazones.
The nitro groups on these hydrazones can be readily reduced using a
variety of reducing agents such as NaBH4/Pd, Pd/H2 and stannous
chloride to produce the corresponding amines. The resulting
aromatic amines can be subjected to reductive amination with a
variety of aliphatic or aromatic aldehydes, such as acetaldehyde,
linear chain aldehydes, branched chain alkyl aldehydes and
benzaldehyde.
Example 4--Efficacy of OF-.DELTA.xyl Analogues for Treating VVC
[0117] Six to eight-week-old BALB/c mice would be intravaginally
infected with C. albicans and dosed once per day with OF-.DELTA.xyl
analogues for two to six days, particularly, three days. The
OF-.DELTA.xyl analogue treated groups would be compared to a
vehicle control group. Several groups of mice would be treated with
various concentrations of OF-.DELTA.xyl analogues. The
occidiofungin treated groups would be expected to reduce fungal
load by more than two logs compared to control groups and
significantly better than naturally occurring occidiofungins. The
mice would be examined for outward signs of distress or irritation.
No behavioral changes including sluggishness, stretching, or
reluctance to consume food is expected. Furthermore, no vaginal
bleeding or swelling is expected following OF-.DELTA.xyl analogue
treatment.
[0118] The murine model of VVC has been reported by Yano et al. A
variation of this method would be followed. Several groups of six
mice would be used to evaluate several concentrations of
OF-.DELTA.xyl analogue and vehicle control. Briefly, six to
eight-week-old BALB/c mice would be treated subcutaneously with 200
ng per mouse of .beta.-Estradiol 17-valerate three days prior to
inoculation with C. albicans (D-3). A subcutaneous dose of
estradiol would be administered every three days (D0, D3) until the
end of the experiment to induce pseudo-estrus. Approximately a 20
.mu.L intravaginal inoculation of a 2.5.times.10.sup.6 colony
forming units (CFU)/mL of C. albicans would define day zero (D0) of
the VVC study. On the same day of inoculation (D0), another
subcutaneous injection of estradiol would be made. OF-.DELTA.xyl
analogues at several concentrations would be suspended in 20 .mu.L
of warm 0.3% Noble agar before intravaginal inoculation. Drug
treatment would be done on day 2 (D2), day 3 (D3) and day 4 (D4) of
the study. On day 5 (D5), the vaginal lumen would be lavaged with
100 .mu.L of sterile PBS with a 200 .mu.L pipette tip. Serial
dilutions and total colony forming units per vaginal lavage would
be determined by plating on YPD plates containing 50 .mu.g/mL of
chloramphenicol. The colony forming units (CFUs) obtained from each
lavage would be counted on plates containing 30-300 colonies for
determining the CFU/mL estimates. Body weight, signs of vaginal
irritation such as swelling or bleeding and clinical signs of
discomfort (stereotypical stretching behavior) would be monitored.
Statistical analyses (T-test) would be done to compare the control
group to treated groups and to compare differences between treated
groups.
Example 5--Methods of Treating Fungal Infections
[0119] Intraperitoneal (ip) and subcutaneous routes of
administration: Toxicological analysis of occidiofungin was carried
out using a suspension of occidiofungin in solvents such as
phosphate buffered saline (PBS), PBS containing 0.5%
methylcellulose and 0.1% Tween 20. Occidiofungin was not completely
soluble in these solvents and suspended particles were observed in
the solution. Sonication mitigated the amount of suspended
particles but not entirely. Occidiofungin was administered to
female C57BI/6 C3H F1 (B6C3F1) mice at age of 6 to 8 weeks via
multiple routes and different doses to estimate toxicity.
[0120] Occidiofungin was administered by intraperitoneal injection
in a single dose at 1, 5, 10, or 20 mg/kg in PBS of body weight.
The compound was also administered in 1 dose at 10 mg/kg in
sterilized 0.5% methylcellulose (constituted with 0.1% Tween-80 in
PBS to promote solubility of test agent) by ip or subcutaneous
injection. The excipient control in each experiment matched the
vehicle. Control groups received sterile PBS or 0.5%
methylcellulose with 0.1% Tween-80 in PBS. Body weight and clinical
signs (movement, posture, skin lesions, appearance of fur
indicating normal grooming, and behaviors) were recorded daily for
5 days. Necropsies were performed on study day 1 or study day 5
following the administration of occidiofungin. Blood and tissue
samples were collected from animals dosed at 10 mg/kg with
occidiofungin in 0.5% methylcellulose. One group of samples was
collected on study day 1 following a single dose by ip injection.
Another group was collected after 5-day observation with the same
single dose through subcutaneous injection. Mice were anesthetized
with isofluorane. Blood was then taken from the retroorbital plexus
for serum biochemistry assays (alkaline phosphatase, alanine
aminotransferase [ALT], aspartate aminotransferase[AST],
gamma-glutamyl transferase, creatine kinase, blood urea nitrogen,
creatinine) and hematology (white blood cell count and white blood
cell differentiation). Body weight was measured immediately after
mice were fully anesthetized and organs (spleen, thymus, brain,
kidney, lung, and liver) were weighed and fixed in 10% neutral
buffered formalin. Histological examination was performed on a
portion of each organ by using routine paraffin-embedding technique
and staining with hematoxylin and eosin (H & E).
[0121] Occidiofungin was also evaluated in a 5-day toxicity study
by using 5 mice per group. Occidiofungin was dissolved in sterile
PBS at dose of 2 mg/kg and administrated by ip injection daily for
5 consecutive days. The control group received sterile PBS. Body
weight and clinical signs were recorded daily. Necropsies were
performed on day 5 following the first administration of
occidiofungin. Clinical chemistry, hematology (the same parameters
as in the single-dose study), and histology (the same organs as in
the single-dose study, except thymus) were evaluated. Histological
sections were reviewed in the same manner as described above. Body
weight, body weight change, organ weight, serum chemistry, and
hematology data were analyzed by T test or 2-way analysis of
variance (ANOVA) followed by Bonferroni posttest using Prism
GraphPad software (San Diego, Calif.). All the analyses were
2-sided, with P<0.05 considered statistically significant.
[0122] Results of intraperitoneal (ip) and subcutaneous routes of
administration are as follows. The body weight differences ranged
from 2% to 12% on the first day after treatment at various doses of
occidiofungin in PBS (containing 0.5% methylcellulose or 0.2% Tween
20). At the end of the studies, it was clear that higher dose
induced more body weight loss, and this change was dose responsive.
However, this effect tended to diminish after the dosing ended,
with a consequent regain of body weight during the following days.
Daily dosing at 2 mg/kg for 5 days had a similar effect on day 5 as
a single 5 mg/kg ip dose. Further, a decrease in absolute total
white blood cell count in mice 1 day after ip administration of
occidiofungin at 10 mg/kg was observed. There were no statistical
differences in hematological or serum chemistry values 5 days after
subcutaneous administration of occidiofungin at 10 mg/kg (Table
4).
TABLE-US-00004 TABLE 4 Hematology and serum chemistry in single
dose experiments using occidiofungin in PBS. Single Dose of
Occidiofungin 0 mg/kg 10 mg/kg A: Hematology WBC (1000/.mu.l) 3.5
.+-. 0.3 1.9 .+-. 0.3 * NEU (%) 16.3 .+-. 2.8 11.6 .+-. 1.4 LYM (%)
77.5 .+-. 3.7 80.4 .+-. 2.2 MONO (%) 3.3 .+-. 0.7 6.8 .+-. 1.7
Serum Biochemistry ALP (U/l) 127.0 .+-. 2.0 67.0 .+-. 7.7 * ALT
(U/l) 59.0 .+-. 5.6 58.0 .+-. 2.6 AST (U/l) 110.0 .+-. 13.3 160.0
.+-. 26.2 GGT (U/l) 20.0 .+-. 2.7 20.0 .+-. 1.6 CK (U/l) 146.3 .+-.
36.4 203.0 .+-. 31.6 BUN (mg/dl) 23.0 .+-. 1.2 21.0 .+-. 1.0 CREAT
(mg/dl) 0.1 .+-. 0.1 0.1 .+-. 0.1 Effect of occidiofungin on
hematology and serum chemistry. Animals were sacrificed the day
after i.p. administration of occidiofungin. * Signifies
statistically significant differences between treated and control
group. B: Hematology WBC (1000/.mu.l) 4.1 .+-. 0.7 5.0 .+-. 1.4 NEU
(%) 18.1 .+-. 2.6 30.8 .+-. 10.8 LYM (%) 73.8 .+-. 4.0 62.5 .+-.
12.7 MONO (%) 5.4 .+-. 2.5 4.6 .+-. 2.0 Serum Biochemistry ALP
(U/L) 81.5 .+-. 17.3 75.6 .+-. 11.7 ALT (U/L) 100.0 .+-. 44.6 20.4
.+-. 4.4 AST (U/L) 95.5 .+-. 37.1 69.4 .+-. 12.0 GGT (U/L) 8.0 .+-.
0.8 7.2 .+-. 2.3 CK (U/l) 154.0 .+-. 44.0 140.8 .+-. 30.4 BUN
(mg/dL) 16.0 .+-. 0.8 13.6 .+-. 1.0 CREAT (mg/dL) 0.25 .+-. 0.05
0.20 .+-. 0.09 Effect of occidiofungin on hematology and serum
chemistry. Animals were observed before sacrificing on day 5 after
subcutaneous administration of occidiofungin.
[0123] In the repeat-dose study, white blood cell differential
counts showed an increase in neutrophils and decrease in
lymphocytes following a 5-day ip administration of occidiofungin at
2 mg/kg (Table 5).
TABLE-US-00005 TABLE 5 Hematology and serum chemistry in 5 day
repeat dose experiment. Single Dose of Occidiofungin 0 mg/kg/day 2
mg/kg/day Hematology WBC (K/ul) 3.5 .+-. 1.0 5.6 .+-. 0.4 NEU (%)
18.0 .+-. 3.3 45.9 .+-. 1.9* LYM (%) 75.8 .+-. 3.9 46.4 .+-. 0.9*
MONO (%) 3.6 .+-. 0.7 4.8 .+-. 1.1 Serum Biochemistry ALP (U/l)
96.4 .+-. 9.5 39.6 .+-. 4.2* ALT (U/l) 49.8 .+-. 7.8 48.0 .+-. 6.5
AST (U/l) 104.0 .+-. 22.0 201.8 .+-. 59.6 GGT (U/l) 19.0 .+-. 6.0
15.2 .+-. 2.9 CK (U/l) 401.8 .+-. 123.9 401.0 .+-. 114.4 BUN
(mg/dl) 24.4 .+-. 4.3 18.0 .+-. 2.8 CREAT (mg/dl) 0.38 .+-. 0.07
0.18 .+-. 0.09 Effect of occidiofungin on hematology and serum
chemistry. Animals were observed before sacrificing on day 5 after
i.p administration of occidiofungin. *Signifies statistically
significant differences between treated and control group.
[0124] Although there were some statistically significant
differences in serum clinical chemistry parameters, no consistent
dose-response effects were noted, which suggests that the
significant effects represented normal biological variation or
experimental effects are not directly related to the action of the
test compound. Generally, no macroscopic findings were observed by
histological examination. No observable differences were present in
the microscopic cell morphology or macroscopic tissue morphology of
brain, liver, lung, thymus, or kidney. One finding was a decrease
in activated thymocytes in the medulla in the 10 mg/kg single-dose
toxicity study. This result in conjunction with decreased thymus
weight or increased neutrophil percentages suggests that
occidiofungin causes a nonspecific stress response. Another likely
explanation is that there is an allergic response to the presence
of the xylose on occidiofungin causing an increase in neutrophils.
In summary, there was no clear evidence for organ-specific
histological effects of occidiofungin.
[0125] Intravenous (iv) administration of occidiofungin Six to
eight-week-old female BALB/c mice (5 mice per group) were given
occidiofungin dissolved in 1.5% hydroxy propyl-beta-cylcodextrin
suspended in phosphate buffered saline (PBS). Spectrometric
inspection at O.D.600 following addition of vehicle to the purified
dried drug had negligible absorbance difference to vehicle without
drug, suggesting that the drug went into solution. For the
experiments, occidiofungin was administered by intravenous (i.v.)
injection into the tail vein at a single dose at 5 mg/kg of body
weight. The excipient control in each experiment matched the
vehicle. Body weight and clinical signs (movement, posture, skin
lesions, appearance of fur indicating normal grooming, and
behaviors) were recorded following administration at one, four,
eight, sixteen, and twenty-four hours. Necropsies were performed at
24 hours following administration of occidiofungin. Blood and
tissue samples from animals dosed at 5 mg/kg of body weight with
occidiofungin in 1.5% hydroxy propyl-beta-cylcodextrin suspended in
PBS were taken 24 hours following excipient or drug administration.
Mice were anesthetized with isofluorane. Blood was then taken from
the retroorbital plexus or heart puncture for serum biochemistry
assays (alkaline phosphatase, alanine aminotransferase, aspartate
aminotransferase, albumin, and blood urea nitrogen) and hematology
(white blood cell count and white blood cell differentiation). Body
weight was measured immediately before treatment and 24 hours later
before the mice were fully anesthetized and fixed in 10% neutral
buffered formalin. Histological examination was performed on a
portion of each organ by using routine paraffin embedding technique
and staining with hematoxylin and eosin (H & E).
[0126] Results of intravenous (iv) administration of occidiofungin
are as follows. Following administration of occidiofungin
intravenously, mice weight ranged between 0% to 21% body
weight-loss 24 hours after treatment and had an average weight loss
of 6.2%. Excipient treated mice body weight showed 8% to 13% body
weight gain 24 hours after treatment and had a 9.6% average
increase in body weight. A consistent behavioral response was
observed at 1 hour and to a lesser extent at 4 hours post i.v.
administration, in which the mice were more lethargic than
excipient treated mice and had ruffled fur. They were responsive to
touch but would move slower than excipient treated mice. Treatment
was not associated with more typical rodent behaviors associated
with severe pain (e.g., writhing, vocalization, or lack of
spontaneous locomotion). No other behavioral signs were observed.
Mice behavior appeared to be normal by 8, 16, and 24 hour post
injection. Generally, no macroscopic findings were observed by
histological examination performed on tissues removed from the
euthanized mice at 48 hours post injection. No observable
differences were present in the microscopic cell morphology or
macroscopic tissue morphology of esophagus, stomach, small
intestine, colon, liver, pancreas, spleen, kidneys, lungs, heart
and brain. Albumin and blood urea nitrogen (BUN) tests were similar
for occidiofungin treated and excipient treated mice (Table 6).
These blood tests are indicative of normal kidney and liver
function. In addition, alkaline phosphatase (ALP) tests were
similar between drug and excipient treated mice (Table 6). These
results indicate normal liver and bone cell function. Normally
aspartate amino transferase (AST) and alanine aminotransferase
(ALT) tests are performed in combination with ALP to assess liver
function. Elevated levels of AST and ALT do suggest heart or liver
damage, but do not necessarily indicate severe organ damage.
Generally, a ratio of AST to ALT less than one is indicative of
liver damage. The ratio in all treated mice was greater than one,
suggesting that the liver is not damaged. AST values ranged from
177 to 1528 (U/1) with a mean value of 765 (U/1) and the ALT values
ranged from 144 to 1273 (U/1) with a mean value of 521 (U/1) (Table
6). Given the variability in AST and ALT levels in treated mice,
only the ALT levels were statistically significant. White blood
cell (WBC) counts were not statistically different between treated
and untreated mice. This suggests that occidiofungin i.v.
administration at 5 mg/kg had no cytotoxicological effect on blood
cells or bone marrow. The absence of elevated levels further
suggests normal spleen function. The percentage of neutrophils was
statistically different in drug and excipient treated mice, while
the percentages of lymphocytes was not statistically different
(Table 6).
TABLE-US-00006 TABLE 6 Percent body weight change, serum chemistry
and hematology following intravenous administration of drug and
excipient control. Single IV Dose of Occidiofungin 5 mg/kg 0 mg/kg
*Weight Change % -6.2 .+-. 9.9 +9.6 .+-. 1.9 Serum Biochemistry
Albumin (g/dl) 3.1 .+-. 0.6 3.6 .+-. 0.2 BUN (mg/dl) 29.6 .+-. 5
24.5 .+-. 3.3 ALP (U/l) 99 .+-. 34 116 .+-. 17 AST (SGOT) U/l 765
.+-. 692 165 .+-. 81 *ALT (SGPT) U/l 521 .+-. 652 36 .+-. 10
Hematology WBC estimate 4750 .+-. 1225 5830 .+-. 1550 *Neutrophils
% 43 .+-. 14 16 .+-. 5 Lymphocytes % 54 .+-. 15 83 .+-. 7 Platelet
estimate (xK/ul) 39 .+-. 21 58 .+-. 58 Animals were sacrificed 24
hours following i.v. administration of occidiofungin. *Signifies
statistically significant differences between treated and control
group. p < 0.05 was considered statistically significant.
[0127] The increase in neutrophils is likely attributed to an
allergic response due to the presence of the xylose on
occidiofungin. There was no statistical difference in platelet
counts between drug and excipient treated mice, suggesting that
occidiofungin does not affect platelet production by the bone
marrow or destroy circulating platelets. The increase in
neutrophils was not observed in mice treated with OF-.DELTA.xyl
(Table 7). In summary, there was no evidence for organ specific
histological effects of occidiofungin. There were no apparent
undesirable effects observed in the serum clinical chemistry and
hematology parameters that would preclude additional animal testing
of the compound. However, the increase in neutrophils does suggest
a possible allergic response that appears to be alleviated with the
removal of xylose.
TABLE-US-00007 TABLE 7 Hematology following intravenous
administration of occidiofungin and OF-.DELTA.xyl drug. Single IV
Dose of Occidiofungin or OF-.DELTA.xyl (5 mg/kg) Hematology
*Neutrophils % 26 .+-. 8.4 5 .+-. 0.7 Lymphocytes % 72 .+-. 9.2 95
.+-. 0.7
[0128] Treatment of Vulvovaginal Candidiasis
[0129] Current antifungal treatment options are plagued with
rapidly increasing occurrence of resistance, high degree of
toxicity and a limited spectrum of activity. Novel antifungal
agents with a unique target, wider spectrum of activity, and
reduced toxicity to the host are desired. This example
characterizes occidiofungin produced by Burkholderia contaminans
MS14. The cellular target of the occidiofungin was determined to be
actin. Actin binding metabolites are generally characterized by
their ability to inhibit polymerization or depolymerization of
actin filaments, which presumably accounts for their severe
toxicity. Occidiofungin, instead, has a subtler effect on actin
dynamics that triggers apoptotic cell death. The efficacy of the
antifungal is demonstrated in treating a vulvovaginal yeast
infection in a murine model.
[0130] Occidiofungin has a wide spectrum of activity against
filamentous and non-filamentous fungi and minimal toxicity in an
animal system. The mechanism of action of occidiofungin differs
from the primary mode of action of the three common classes of
antifungals. Occidiofungin has been observed to rapidly induce
apoptosis in yeast cells at the minimal inhibitory concentrations.
The primary cellular target of occidiofungin is actin.
Actin-mediated cellular processes in yeast, such as endocytosis,
nuclear segregation and hyphal formation, were all disrupted
following addition of subinhibitory concentrations of
occidiofungin. Occidiofungin's binding to actin interferes with
F-actin filament cable stability and does not interfere with
polymerization or depolymerization of actin filaments.
Occidiofungin binds to F-actin with an estimated dissociation
constant (Kd) of 1000 nM and has a high saturation of binding ratio
of more than twenty molecules of occidiofungin to one actin
monomer. Given the high binding ratio, it was predicted that the
microscopic Kd value (which captures the affinity of one
occidiofungin molecule binding to actin) is significantly lower
than the 1000 nM dissociation constant.
[0131] In the repeat-dose toxicity study, the hematology tests
revealed that white blood cell differential counts showed an
increase in neutrophils and decrease in lymphocytes. Neutrophils
play important roles in host defense against all classes of
infectious agents and pathology of various inflammatory conditions.
The increase in neutrophils also suggests a possible inflammatory
or mild stress response. A potential reason for the observed
response is the presence of a xylose moiety on the novel amino acid
2 (NAA2) or the NAA2 residue itself (FIG. 1). Mammalian immune
system can recognize N-glycans containing .beta.-(1,2)-xylose and
.alpha.-(1,3)-fucose residues. Novel analogs at the NAA2 position
of occidiofungin were synthesized to test whether the xylose on
NAA2 residue is responsible for the development inflammation
response.
[0132] In addition, occidiofungin has sub-micromolar activity
against Pythium species which lacks ergosterol in the membrane and
against Cryptococcus neoformans which is resistant to
echinocandins. Preliminary toxicological analyses of occidiofungin
using a murine model indicated that it was well tolerated at
concentrations of 10 to 20 mg/kg, but it demonstrates a mild
allergic or stress response. Blood chemistry analyses and
histopathology performed on multiple organs showed a transient
non-specific stress response with no damage to organ tissues. These
data suggest that occidiofungin is a promising candidate for
development as a clinically useful antifungal agent.
[0133] Spectrum of activity of occidiofungin against clinically
relevant fungi Occidiofungin causes cell death in fungi through a
mechanism of action that is distinct from the clinically used
classes of antifungals. Due to its unique mechanism of action,
occidiofungin has sub-micromolar activity against azole and
echinocandin resistant strains of fungi. Strains of Candida
albicans, Candida glabrata, and Candida parapsilosis that were
resistant to fluconazole and caspofungin were sensitive to
occidiofungin (Table 8). Non-albicans strains are believed to be
the primary cause of recurrent vulvovaginal candidiasis.
Furthermore, strains of Candida parapsilosis and C. neoformans that
were resistant to treatment with caspofungin were found to be
susceptible to treatment with occidiofungin. Occidiofungin was also
found to have a broader spectrum of activity than clinically
available antifungals and was found to be active against
Aspergillus, Mucor, Fusarium, and Rhizopus species. Several strains
of the dermatophyte Trichophyton were found to also be susceptible
to occidiofungin treatment, including azole and terbinafine
resistant strains. A summary of the results, as reported in Table
8, indicate that occidiofungin has activity against filamentous and
non-filamentous fungi at sub-micromolar concentrations and has a
broader spectrum of activity compared to other clinically available
antifungals. Furthermore, sensitivity of fungal strains resistant
to azoles and echinocandin class of antifungals, support the notion
that occidiofungin is functioning via a novel mechanism of
action.
[0134] Occidiofungin has sub-micromolar activity against azole and
echinocandin resistant strains of fungi. Susceptibility of multiple
strains of each species (Filamentous fungi: Aspergillus fumigatus,
Aspergillus flavus, Fusarium sp. (including solani and oxysporum),
Mucor sp., Rhizopus sp., Trichophyton rubrum, and Trichophyton
mentagrophytes; and Yeasts: C. albicans, C. glabrata, C. krusei, C.
parapsilosis, C. tropicalis, and Cryptococcus neoformans) to
occidiofungin was tested and antibiotic resistant strains were used
when available (Table 8). Fluconazole resistant C. albicans and C.
glabrata were sensitive to occidiofungin. Inhibitory concentrations
of occidiofungin to these isolates were .ltoreq.2 .mu.g/mL, while
the MIC of fluconazole against these strains was .gtoreq.32
.mu.g/mL. Rhizopus spp. and C. neoformans were more sensitive to
occidiofungin than fluconazole. Cryptococcus neoformans is
insensitive to echinocandins, but is susceptible to occidiofungin
at sub-micromolar concentrations.
[0135] Minimum inhibitory concentration (MIC) susceptibility
testing was performed according to the CLSI M27-A3 and M38-A2
standards for the susceptibility testing of yeasts and filamentous
fungi, respectively. Incubation temperature was at 35.degree. C.
and the inoculum size was 0.5-2.5.times.10.sup.3 colony-forming
units (CFU)/mL and 0.4-5.times.10.sup.4 conidia/mL for yeasts and
filamentous fungi, respectively. Inoculum concentration for
dermatophytes was 1-3.times.10.sup.3 conidia/mL. RPMI was used
throughout as the growth medium and Cryptococcus strains were
tested in YNB. Occidiofungin MICs were recorded at 50% and 100%
growth inhibition after 24 and 48 hours of incubation, with the
exception of dermatophytes which were incubated for 96 hours.
Fluconazole MICs against Candida strains were recorded at 50%
inhibition after 24 hours and against Cryptococcus strains after 72
hours. Voriconazole MICs were recorded at 100% inhibition after 24
hours for zygomycetes and after 48 hours for Fusarium and
Aspergillus strains. Voriconazole MICs were recorded at 80%
inhibition after 96 hours of incubation for dermatophytes. S.
cerevisiae deletion mutants were obtained from the commercially
available BY4741 deletion library (Thermo Scientific).
Susceptibility testing was carried out on inoculum size of
0.5-1.times.10.sup.4 cells/ml in YPD media at 30.degree. C. and
MICs recorded after 48 and 60 hours.
[0136] Development of Occidiofungin as a Treatment for VVC
[0137] The work that is being conducted will lead to a new,
first-in-class anti-infective chemotherapeutic targeted
specifically for fungal infections. Fungal pathogens infect more
than 7,000,000 adults in the US each year and are a significant
source of economic health cost for society. The lead molecule
occidiofungin serves as a platform for development of t therapies
to treat VVC.
[0138] In the United States it is estimated that seventy percent of
the female population will suffer from VVC. A subset of this
population, nearly 5,000,000 women annually, suffer from RVVC.
RVVC's estimated health care cost in the USA is $4-5 billion per
year. This estimate is based on increased medical costs to treat
VVC and the economic impact from lost productivity. The global cost
of VVC and RVVC is expected to be over 10 billion per year. VVC and
RVVC also place a significant cost on quality of life for any
infected individual, their family, and employers. Although there
are several preventative measures in development, there is no
effective therapeutic. An effective therapeutic that could treat
VVC in a relatively non-invasive methodology could significantly
reduce the cost to patients, while at the same time increase their
quality of life. Overall, a new therapeutic would reduce the impact
of these infections on the economy. Additionally, there is a
growing concern over the increase in drug resistant strains of
Candida and the lack of new treatment of options available. These
resistant strains are expected to increase the global costs
associated with RVVC.
[0139] Occidiofungin is superior in clinical setting as an
antifungal for several reasons. For example, its antifungal
activity has been demonstrated against a wide array of fungi,
including those resistant to current antifungals in use. MICs of
occidiofungin against Candida species are between 0.5 and 2.0
.mu.g/mL, which is similar in activity to echinocandins and
amphotericin B. Pharmacodynamic (Time Kill) experiments revealed
that occidiofungin is rapidly fungicidal against Candida albicans,
which is better than current clinically approved antifungals.
Occidiofungin retains its in vitro potency in the presence of 5%
and 50% human serum with a minimum lethal concentration (MLC) of 2
and 4 .mu.g/mL, respectively. An alternative target for
occidiofungin other than targeting ergosterol production, binding
to ergosterol, or inhibiting the 1,3-.beta.-glucan synthase enzyme,
is advantageous because these mechanisms are prone to resistance.
In vivo toxicity studies show that body and organ weight changes
induced by occidiofungin are not associated with any negative gross
or microscopic findings. Hematology and serum biochemistry tests
reveal that occidiofungin does not significantly alter functions of
organs. Occidiofungin demonstrates a significant reduction in
fungal load in murine VVC and system yeast infection models.
[0140] These factors provide occidiofungin with unique advantages
that are necessary to be an effective treatment for VVC and
RVVC.
[0141] Efficacy of Occidiofungin in Treating a Murine Model of
VVC
[0142] Six to eight-week-old BALB/c mice were intravaginally
infected with C. albicans and dosed once per day with occidiofungin
for three days. The occidiofungin treated groups were compared to a
vehicle control group. Three groups of six mice were treated with
100, 50, and 0 .mu.g of occidiofungin suspended in 0.3% Noble agar.
The occidiofungin treated groups reduced fungal load by more than
two logs (FIG. 7). The reduction in fungal load with both treatment
groups was statistically significant from vehicle control
(p<0.001). There was no statistically significant difference
between the treated groups (p=0.33), suggesting that the lower
limit of occidiofungin dosing was not achieved in the experiment.
During the course of the study, the mice were examined for outward
signs of distress or irritation. No behavioral changes including
sluggishness, stretching, or reluctance to consume food was
observed. Furthermore, no vaginal bleeding or swelling was observed
following treatment.
[0143] The murine model of VVC has been reported by Yano et al. A
variation of this method was followed. Three groups of six mice
were used to evaluate two concentrations of occidiofungin (100
.mu.g and 50 .mu.g) and vehicle control (0.3% Noble agar). Briefly,
six to eight-week-old BALB/c mice were treated subcutaneously with
200 ng per mouse of .beta.-Estradiol 17-valerate three days prior
to inoculation with C. albicans (D-3). A subcutaneous dose of
estradiol was administered every three days (D0, D3) until the end
of the experiment to induce pseudo-estrus. Approximately a 20 .mu.L
intravaginal inoculation of a 2.5.times.10.sup.6 colony forming
units (CFU)/mL of C. albicans defines day zero (D0) of the VVC
study. On the same day of inoculation (D0), another subcutaneous
injection of estradiol was made. Lyophilized powder of
occidiofungin containing either 100 .mu.g or 50 .mu.g of
occidiofungin was suspended in 20 .mu.L of warm 0.3% Noble agar
before intravaginal inoculation. Drug treatment was done on day 2
(D2), day 3 (D3) and day 4 (D4) of the study. On day 5 (D5), the
vaginal lumen was lavaged with 100 .mu.L of sterile PBS with a 200
.mu.L pipette tip. Serial dilutions and total colony forming units
per vaginal lavage were determined by plating on YPD plates
containing 50 .mu.g/mL of chloramphenicol. The colony forming units
(CFUs) obtained from each lavage were counted on plates containing
30-300 colonies for determining the CFU/mL estimates. Body weight,
signs of vaginal irritation such as swelling or bleeding and
clinical signs of discomfort (stereotypical stretching behavior)
were monitored. Statistical analyses (T-test) were done to compare
the control group to treated groups and to compare differences
between treated groups. All the analyses were 2-sided, with
P<0.05 considered statistically significant.
TABLE-US-00008 TABLE 8 Activity of occidiofungin against
filamentous and non-filamentous fungi. Voricona- Flucona-
Occidiofungin (.mu.g/mL) zole zole 24 hours 48 hours 72 hours 96
hours MIC MIC Species 50% 100% 50% 100% 50% 100% 80% 100%
(.mu.g/mL) (.mu.g/mL) *Trichophyton 1 2 0.25 >16 mentagrophytes
10207 Trichophyton 1 2 0.06 >16 mentagrophytes 28556
Trichophyton 1 2 0.06 16 mentagrophytes 28641
.sup.&Trichophyton 1 2 0.008 0.25 rubrum 11199 Trichophyton 1 2
0.03 2 rubrum 28658 Trichophyton 1 2 0.03 2 rubrum 28659 Rhizopus 4
8 -- 8 16 microsporus 28506 Rhizopus 4 8 -- 8 >16 oryzae 28403
Rhizopus 2 4 -- 8 >16 microsporus 27785 Mucor 4 8 4 8 >16
circinelloides 19445 Mucor 2 4 -- 4 >16 racemosus 27784 Mucor 2
4 -- 4 >16 fragdis 27782 Fusarium 2 4 -- 4 >16 solani 28386
Fusarium 2 4 -- 4 >16 oxysporum 27718 Fusarium 2 4 2 4 >16
solani 18749 Aspergillus -- 4 -- 4 1 flavus 28517 Aspergillus 2 4
-- 4 2 flavus 28455 Aspergillus 2 4 -- 4 2 flavus 28445 Aspergillus
-- 4 -- 4 1 fumigatus 28434 Aspergillus -- 2 -- 2 1 fumigatus 28435
Aspergillus 2 4 2 4 1 fumigatus 28436 .sup.#Candida -- 1 -- 2 32
albicans 23512 Candida 4 8 4 8 8 albicans 28200 Candida -- 2 -- 2
0.125 albicans 28102 .sup.#Candida 2 4 -- 4 64 glabrata 27243
Candida -- 2 -- 2 4 glabrata 25742 Candida 4 8 4 8 >64 glabrata
28271 Candida 2 4 -- 4 16 krusei 9541 .sup.#Candida 4 8 4 8 64
krusei 28415 Candida 4 8 4 8 16 krusei 28570 .sup.+Candida 2 4 -- 4
0.125 parapsilosis 2006 Candida 4 8 4 8 0.25 parapsilosis 28364
Candida -- 4 -- 4 0.25 parapsilosis 28174 Candida -- 2 -- 2 0.25
tropicalis 9624 Candida 4 8 4 8 0.125 tropicalis 28272 Candida 4 8
4 8 0.125 tropicalis 28478 .sup.+Cryptococcus -- 2 4 neoformans
19526 Cryptococcus -- 2 2 neoformans 27708 Cryptococcus -- 1 4
neoformans 28446
Example 6--Actin Binding Properties of Occidiofungin Analogs
[0144] To characterize the actin binding properties of the
dodecylamine and dihydrosphingosine analogs of occidiofungin, a
co-sedimentation assay was performed. Native occidiofungin had a Kd
value of 1000 nM and a saturation of binding ratio of .about.25:1
(ligand:actin monomer, FIG. 9A). Further, phalloidin had an
estimated Kd value of .about.8 nM with a saturation of binding
ratio of .about.0.7:1 (ligand:actin monomer, FIG. 9C). The
dodecylamine analog was determined to have a Kd value of 4200 nM
with a saturation of binding ratio of .about.34:1 (ligand:actin
monomer) (FIG. 9B). Interestingly, the dihydrosphingosine analog
had a Kd value of 25 nM with a saturation of binding ratio of
.about.1.8:1 (ligand:actin monomer, FIG. 9D). The dodecylamine
analog had a similar saturation of binding to actin as the native
compound with approximately a 4-fold lower binding affinity to
actin. The dihydrosphingosine analog showed an actin binding
property closer to phalloidin's and had a 13.5-fold lower
saturation of binding ratio than native occidiofungin. The decrease
in dissociation constant is likely attributed to the low saturation
of binding for the dihydrosphingosine analog compared to native
occidiofungin and not an increase in affinity to actin.
[0145] Given that the actin binding properties of the
dihydrosphingosine analog were different from native occidiofungin,
the analog was tested to determine whether its mechanism of actin
binding was different than native occidiofungin. Native
occidiofungin does not interfere with actin polymerization or
depolymerization, but rather interferes with actin cable formation
triggering ROS accumulation and apoptotic cell death. The molar
ratio of occidiofungin, analogs of occidiofungin, or phalloidin to
actin in the polymerization and depolymerization assay was close to
1:1. The pyrene fluorescence readings approximately one hour after
incubation in the polymerization buffer plateaued at around 19,000
for phalloidin and 21,000 AU for the dihydrosphingosine analog and
native occidiofungin (FIG. 10). Following incubation in the
depolymerization buffer, actin filaments incubated with phalloidin
had pyrene fluorescent readings above 5,000 AU, while actin
filaments incubated with dihydrosphingosine analog and native
occidiofungin had similar readings as no drug control at 2,500 AU.
Phalloidin completely blocked the depolymerization of F-actin. The
dihydrosphingosine analog did not interfere with polymerization or
depolymerization of actin, similar to what was observed with native
compound (FIG. 10). The lack of any interference with the
polymerization or depolymerization of actin by the
dihydrosphingosine analog supports the assumption that the
dihydrosphingosine analog and native occidiofungin have the same
binding region on actin.
Example 7--Improved Analogs of Occidiofungin
[0146] This example provides strategies to produce novel analogs of
occidiofungin. Oxidative cleavage of the diol group present in the
aliphatic chain of NAA2 followed by reductive aminations of alkyl
amines restored the aliphatic structure of the native compound. The
methods described to synthesize the analogs of occidiofungin
provide an efficient strategy for generating novel analogs of
occidiofungin. One of the analogs synthesized was identified to
have reduced antifungal activity but significantly lower
dissociation constant and stoichiometry in an actin
co-sedimentation study. These results showed that the fatty acid
moiety in native occidiofungin plays an important role with regards
to its antifungal activity and actin binding properties.
[0147] Occidiofungin needs to enter the cell to reach its cellular
target actin. Against S. cerevisiae, the undecylamine and
dodecylamine analogs were both inactive at a concentration that was
sixty-four-fold higher than native compound, while the dodecylamine
analog only had a four-fold reduction in affinity for actin. The
lack of activity of undecylamine and dodecylamine analogs suggests
that the hydroxyl groups on the side chain may be essential for the
uptake of occidiofungin into susceptible cells. This is supported
by the fact that the dodecylamaine analog was capable of binding to
actin in a similar manner as native occidiofungin. Given that the
inhibitory activity of the dihydrosphingosine analog can be
partially restored, the introduction of the hydroxyl groups near
the base of the lipid moiety may aid in cellular entry and enable
the analog to reach the actin target.
[0148] Accordingly, some embodiments of the invention provide
modifying an occidiofungin analog disclosed herein to render it
suitable for entry into the cell. Such modification can be
attachment, for example, via a covalent bond, to a moiety that
facilitates entry of the occidiofungin analog into a cell. Such
modification can also be inclusion of the occidiofungin analogs
disclosed herein into vesicles or capsules that facilitate uptake
of the occidionfungin analog into a cell. The vesicles or capsules
can be lipid vesicles or 1-Palmitoyl-2-oleoylphosphatidylcholine
(POPC) vesicles.
[0149] Intravenous administration of occidiofungin triggered a mild
inflammation or allergic response. Xylose is only reported to be
present in plants and some invertebrates and is not known to be
present in mammals. .beta.-(1,2)-xylose is involved in a common
cross-reactive antigenic determinant (CCD), and is shared by a
variety of glycoproteins recognized by IgE antibodies of patients
with food or respiratory allergies. Both IgE and IgG2 antibodies
responded to CCDs and that CCDs affected both Th1- and Th2-type
responses. .beta.-(1,2)-xylose modifications of N-glycans modify
specific IgE-binding in individuals allergic to Lyc e 2, a
glycosylated allergen of tomato. The xylose free analogs of
occidiofungin disclosed herein provide therapeutically superior
analogs of occidiofungin.
[0150] The dihydrosphingosine analog exhibits low binding constant
and saturation stoichiometry. The high saturation of occidiofungin
to actin (25:1) can be attributed to the self-assembly mechanism of
native occidiofungin monomers following its binding to F-actin.
This is not uncommon activity for lipopeptides and the formation of
self-assembled complexes was observed with several lipopeptide
antibiotics. The polarity of dihydrosphingosine analog is much less
than the native compound (eluted at 33% versus 47% water on HPLC;
FIGS. 5-6), which might contribute to the loss of the self-assembly
mechanism, in the sense that amphiphilicity leads to self-assembly
at high concentrations for peptide amphiphiles. However, the
relationship between self-assembly and bioactivity is complicated
and remains unclear. Reduced antifungal activity of the
dihydrosphingosine analog may be attributed to the reduced polarity
and self-assembly property. The dihydrosphingosine analog had a Kd
value of 25 nM and had a stoichiometry closer to a 1:1
(ligand:actin) binding. The dihydrosphingosine analog may have a
higher affinity for actin. The lower Kd value likely represents the
microscopic dissociation constant (which captures the affinity of
one occidiofungin binding to actin) versus the macroscopic Kd value
captured by the native compound (24:1 ligand:actin).
[0151] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated within
the scope of the invention without limitation thereto.
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Sequence CWU 1
1
117PRTArtificial SequencepeptideMISC_FEATURE(2)..(2)Xaa = 3
amino-5,6-dihydroxy-7-O-xylose- octadecanoic
acidMISC_FEATURE(4)..(4)Xaa = beta-hydroxy tyrosine 1Asn Xaa Ser
Xaa Gly Asn Ser1 5
* * * * *