Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF TREATING CLOSTRIDIUM DIFFICILE-ASSOCIATED
DIARRHEA
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the treatment of a disease
caused by the presence of a bacterium selected from the
group consisting of Clostridium species, Staphylococcus
species, and Enterococcus species and combinations
thereof, in particular a disease caused by the presence of
a bacterium selected from the group
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consisting of Clostridium difficile ("C. difficile"),
Clostridium perfringens ("C.
perfringens"),
Staphylococcus aureus ("S. aureus") and combinations
thereof, more particular a disease caused by the
presence of C. difficile. The disease may be colitis,
pseudomembranous colitis, or diarrhea.
2. Description of the Related Art
[0004]
Antibiotic-associated diarrhea (AAD) is caused
by toxin producing strains of C. difficile, S. aureus
including methicillin-resistant Staphylococcus aureus
(MRSA), and Clostridium perfringens (C. perfringens).
AAD represents a major economic burden to the healthcare
system that is conservatively estimated at $3-6 billion
per year in excess hospital costs in the U.S. alone.
[0005]AAD is a significant problem in hospitals and long-
term care facilities and in the community. C. difficile
is the most common cause of AAD in the hospital setting,
accounting for approximately 20% of cases of AAD and the
majority of cases of antibiotic-associated colitis
(AAC). The rising incidence of C. difficile associated
diarrhea (CDAD) has been attributed to the frequent
prescription of broad-spectrum antibiotics to
hospitalized patients [Wilcox et al., Lancet 1996, 348:
767-.8].
[0006]The most serious form of the disease is
pseudomembranous colitis (PMC), which is manifested
histologically by colitis with mucosal plaques, and
clinically by severe diarrhea, abdominal cramps, and
systemic toxicity. The overall mortality rate from CDAD
is low, but is much greater in patients who develop
severe colitis or systemic toxicity. A recent study has
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shown that even when death is not directly attributable
to C. difficile, the rate of mortality in CDAD patients
as compared to case-matched controls is much greater.
N0071Diarrhea and colitis are caused by the elaboration
of one or more C. difficile toxins. The organism
proliferates in the colon in patients who have been
given broad-spectrum antibiotics or, less commonly,
cancer chemotherapy. CDAD is diagnosed in approximately
20% of hospitalized patients who develop diarrhea after
treatment with such agents.
[0008] Current therapy for AAD or CDAD includes
discontinuation of implicated antimicrobial or
chemotherapy agents, nonspecific supportive measures,
and treatment with antibiotics directed against C.
difficile. The most common antimicrobial treatment
options include vancomycin, and Metronidazole. Treatment
of CDAD with antibiotics is associated with clinical
relapse of the disease. Frequency of relapse is reported
to be 5-50%, with a 20-30% recurrence rate being the
most commonly quoted figure. Relapse occurs with nearly
equal frequency regardless of the drug, dose, or
duration of primary treatment with any of the
antibiotics listed above. The major challenge in therapy
is in the management of patients with multiple relapses,
where antibiotic control is problematic.
[0009]The two most commonly utilized specific therapies
are vancomycin and metronidazole, though vancomycin is
the only drug approved by the FDA for this indication.
However, Vancomycin is not recommended for first-line
treatment of CDAD mainly because it is the only
antibiotic active against some serious life-threatening
multi-drug resistant bacteria. Therefore, in an effort
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to minimize the emergence of vancomycin-resistant
Enterococcus (VRE) or
vancomycin-resistant
Staphylococcus aureus (VRSA), the medical community
discourages the use of this drug except when absolutely
necessary.
[001 ]Metronidazole is recommended as initial therapy out
of concern for the promotion and selection of vancomycin
resistant gut flora, especially enterococci. Despite
reports that the frequency of C. difficile resistance
may be >6% in some countries, metronidazole remains
nearly as effective as vancomycin, is considerably less
expensive, and can be used either orally or
intravenously. Metronidazole is associated with
significant adverse effects including nausea,
neuropathy, leukopenia, seizures, and a toxic reaction
to alcohol. Furthermore, it is not safe for use in
children or pregnant women.
[0011]Although both agents are effective in treating the
infection, increasing rates of treatment failures and
recurrence of diarrhea in approximately 20% of patients
that initially respond are deficiencies of standard
therapies. Therapy with metronidazole has been reported
to be an important risk factor for VRE colonization and
infection. In addition, the current treatment regime is
rather cumbersome, requiring up to 500 mg qid for 10 to
14 days. Thus, there is a need for better treatment for
cases of CDAD as well as for cases of other AAD and AAC.
[0012] Therefore, there is a need to develop a
bactericidal drug with a low propensity to generate
resistance, having reduced or no cross-resistance to
existing antimicrobials and/or a prolonged post-
antibiotic effect.
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Summary of the invention
In a broad aspect, the present invention provides use of a
composition in an amount that is therapeutically effective in
treating a disease or disorder caused by the presence of
Clostridium difficile, comprising no less than 90% by weight of
the compound of formula II:
OH
OH / s
8
R 0 OH
CI
of.'X0 /
/ I \ 0flI
it OH
0
o HO CI
19
OH
and 1% to 5% by weight of the compound of formula IX:
0 0
HO *0.< OH
HO
0
1
0 1 0
0 Ci
OH Me0 OH 14 OH
CH3 CI
IX(OP-1435)
4a
CA 02626698 2012-08-16
and 0.1% to 5% by weight of the compound of formula XIV:
2X:
HO.-1--..õT
0 OH
HO
1
..,%0H IVI
0 0 CH3
GI
= -4> --0
kw"
e0 OH/?-----HO Ali. OH
CI
XIV
XIV (lipiarmucin A4) for treating the disease or disorder caused
by the presence of Clostridium difficile.
In another broad aspect the present invention provides a
pharmaceutical composition comprising no less than 90% by weight
of a compound of formula II.
/ \ h9F1
/ ss
OH
ii /
7
R 0 OH
CI
\
/ \ 00.- .00
0*c
/ 1 0 411 OH
---..") 0
S
..? o HO CI
19
OH '
,
0.1% to 5% by weight of a compound of formula XIV
4b
CA 02626698 2012-08-16
0 0
HO
0 CV(
HO
0
0 I 0 CH3
0 GI
0 0
0H ?vie OH HO OH
CI
(lipiarmycin A4)
and
a 0
HO
.L-( H1)<'
0
1
0 0
ArcCI
0 0 0
---+-01-4 Med 801-1110'.' - 0H
CH3 Ci
IX (OP-1435).
In a further broad aspect, the present invention provides use of
a pharmaceutical composition for treating a disease or disorder
caused by the presence of Clostridium difficile, wherein the
pharmaceutical composition comprises a compound of formula IX
(OP-1435)
4c
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X
0 0
HO
OH
HO
I;
I 0
'---,, =
0 0 0 Ali CI
--1\p--
11111,11
OH PAe I-01 F1
CH3 ,...,
M(01'3-1435).
In a further broad aspect, the present invention provides a
pharmaceutical composition comprising a compound of formula of
formula IX (OP-1435):
aX'0
HO
OH
Ho)ct > ,(N
0
0 0 0 4101 CI
¨
0H meo cH HO OH
CH3 ,...1
IX (0P-1435).
4d
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[0013] The present invention provides a method of
treating a disease or disorder caused by the presence of
a bacterium selected from the group consisting of
Clostridium species, Staphylococcus species,
Enterococcus species and combinations thereof comprising
administering to a patient in need an effective amount
of a mixture. The mixture comprises an effective amount
of tiacumicin B and an additional macrocycle selected
from the group consisting of:
.L
0 0
HO
OH
0
0 0
0 0 Api
0H kle0 OH HO OH
CI
III (OP-1416, RT ratio 0.71),
OH
HO
HOqp--F
Yjc 0
0
0
u
meo I AO OH
CI
IV (OP-1415, RT ratio 0.81),
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0 0
fc10< OH
HO
0 /
O I 0
CI
0 0 0 10
OH WO OH HO OH
V (OP-1417, RT ratio: 0.84),
0X0
HO*0< 0H
HO
0
O 0
o o o CI
OH
0H Me0 OH HO
CH3 CI
IX (OP-1435, RT ratio:1.13),
OXL
0
HO)cCif. OH
HO
0
I ci
O 0 o
OH M ,õe0 OH OH
CI
X (OP-1437, RT ratio:1.19),
0 0
HO:*C OH
HO
0
O 0
O 0--0 ao CI
= Met HO OH
ci
XI (OP-1402, RT ratio:1.24),
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0 0
HO
OH
H0* <
0
0
0
XII (OP-1433, RT ratio: 1.39),
HO
0 o
HO
/ OH
0
0 I
0 0 0
Me0 OH H OH
XIII (OP-1438, RT ratio: 1.48),
HO
O
HO H
0
0
0 CH3
CI
0 0 0
0H Me0 OH HO 41111-17 OH
CI
XIV (lipiarmycin A4, 09-1405, RT ratio: 0.89), and
combinations thereof. When the compound of formula XIV
is present, the mixture comprises about 0.1 to about 5%
compound of formula XIV.
[0014]Preferably, the mixture comprises at least 90%
tiacumicin B by weight. More preferably, the mixture
comprises at least 95% tiacumicin B by weight.
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[0015]Preferably, the mixture comprises at least 1%, more
preferably, from about 2% to about 5%, of additional
macrocycle by weight.
[0016]Preferably, the mixture comprises about 0.1% to
about 5%, more preferably 0.3% to 3%, in particular 0.3%
to 1.5%, especially about 1%, lipiarmycin A4 by weight.
[0017]Preferably, when liapiarmycin A4 is present, the
mixture also comprises at least one of the following
compounds:
7y0
OH
00<
OH
HO
0 /
0 0
io
OH Me0 oHHO OH
CI
VI (OP-1431, tiacumicin F, RT ratio: 0.92),
OH
HO
/0*< OH
0 0
0 CI
LW
OH Mt HO
HO OH
CI
VII (Op-1432, Tiacumicin C, RT ratio:0.95), and
.L
0 0
HO*0
HO
0 /OH
0
0
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VIII (OP-1434, Tiacumicin A, RT ratio:1.10).
[0018] Preferably, the mixture exhibits an HPLC profile
substantially depicted at Figure 5
[0019] Preferably, the disease or disorder treated in
accordance with the present invention is associated with
C. difficile, C. perfringens, S. aureus, and
combinations thereof. More preferably, the disease or
disorder treated in accordance with the present
invention is associated with C. difficile.
[0020] Preferably, the disease treated in accordance with
the present invention is diarrhea or colitis, in
particular diarrhea, more particularly CDAD.
[0021]Preferably, the mixture in accordance with the
present invention is prepared by a process comprising:
culturing a microorganism in a nutrient medium
to accumulate the mixture in the nutrient medium; and
isolating the mixture from the nutrient
medium;
the nutrient medium comprises an adsorbent to
adsorb the mixture.
[0022]The nutrient medium preferably comprises 0.5-15% of
the adsorbent by weight. The absorbent is preferably an
adsorbent resin. More preferably, the adsorbent resin
is selected from the group consisting of Amberlite
XAD16, XAD16HP, XAD2, XAD7HP,XAD1180, XAD1600, IR050,
and Duolite@ XAD761. The
microorganism is preferably
Dactylosporangium aurantiacum subspecies hamdenensis.
The nutrient medium comprises, based on weight, 0.2% to
10% of glucose, 0.02% to 0.5% of K21-1PO4, 0.02% to 0.5% of
MgSO4.7H20, 0.01 % to 0.3% of KC1, 0.1% to 2% of CaCO3,
0.05% to 2% of casamino acid, 0.05% to 2% of yeast
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extract, and 0.5% to 15% of XAD-16 resin. The culturing
step is preferably conducted at a temperature from about
25 to about 35 C and at a pH from about 6.0 to about
8.0-
[0023] Preferably, the disease treated in accordance with
the present invention is associated with the use of
antibiotics or cancer chemotherapies or antiviral
therapy.
[0024]In accordance with one preferred embodiment, the
mixture is administered in an amount of about 50 mg to
1000 mg, more preferably 100 mg to 400 mg, in particular
200 mg, one to three times daily, more preferably once
or twice daily, in particular twice daily, within three
to fifteen days, in particular around ten days. Oral
administration is preferred.
[0025] The treatment of the present invention may allow for
the effective treatment of diarrhea diseases associated
with enterotoxigenic strains of C. difficile, S. aureus,
and C. perffingens without compromising systemic
antibiotics and without increasing vancomycin resistant
enterococci (VRE) in the gut. The present invention also
reduces the presence of VRE in the gut.
[0026] Other objects and features of the present invention
will become apparent from the following detailed
description considered in conjunction with the
accompanying drawings. It is to be understood, however,
that the drawings are designed solely for purposes of
illustration,
It should be further understood that
the drawings are not necessarily drawn to scale and
= that, unless otherwise indicated, they are merely
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intended to conceptually illustrate the structures and
procedures described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] in the drawings:
[0028] Figure 1 shows the Phase 1B-MD Dosing schedule.
[0029]Figure 2 shows the bacteroides count following
treatment. Pairs signed-ranks test, 2 tailed. For counts
< 3 log 10, a value of 2.9 was used.
[0030]Figure 3 shows the effect of Vancomycin therapy vs
B. fragilis group.
[0031] Figure 4 shows the quantitative reduction of C.
difficile vegetative counts after treatment with MCC.
10032]Figure 5 is a typical HPLC profile of the mixture,
which may be used in the method of the present
invention. 7
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED
EMBODIMENTS
[0033] The definitions of certain abbreviations or terms
used in the present application are provided as follows:
AAD = antibiotic-associated diarrhea
ATCC = American Type Culture Collection
C = carbon 13
CO2 carbon dioxide
N2 = nitrogen
H2 = hydrogen
TAPS = N-
Tris(hydroxymethyl)methy1-3-
aminopropanesulfonic acid
MOPS = 3-(N-Morpholino)propanesulfonic acid
CDAD = Clostridium difficile-associated diarrhea
CLSI - Clinical and Laboratory Standards Institute,
formerly NCCLS
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EDH effective dose to produce 50% response
HPLC = high performance liquid chromatography
IR = infrared spectroscopy
LLOQ - lower limit of quantification
MCC - Macrocycle-Containing Composition
MIC = minimum inhibitory concentration
MICH = minimum inhibitory concentration to inhibit
50% of bacterial strains tested
MICH - minimum inhibitory concentration to inhibit
90% of bacterial strains tested
MRSA = methicillin-resistant Staphylococcus aureus
NCCLS - National Commmittee for Clinical Laboratory
Standards, now CLSI
PMC = pseudomembranous colitis
VRE vancomycin-resistant enterococci
VRSA = vancomycin-resistant Staphylococcus aureus
[0034]The term "antibiotic-associated condition" refers
to a condition resulting when antibiotic therapy
disturbs the balance of the microbial flora of the gut,
allowing pathogenic organisms such as enterotoxin
producing strains of C. difficile, S. aureus and C.
perfringens to flourish. These organisms can cause
diarrhea, pseudomembranous colitis, and colitis and are
manifested by diarrhea, urgency, abdominal cramps,
tenesmus, and fever among other symptoms. Diarrhea, when
severe, causes dehydration and the medical complications
associated with dehydration.
[0035]The term "MCC" refers to a preparation primarily
containing tiacumicin B with respect to the whole
antibiotic substance (e.g., at least 90%, preferably 95%-
98% by HPLC assay). MCC also comprise a small amount
(e.g., at least 1%, preferably 2%-5%) of tiacumicin B
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related compounds, i.e., lipiarmycin A4 and at least one
of compound of formula III-XIV shown above. PCT
application PCT/US03/21977, having an international
publication number of WO 2004/014295 A2, provides a
process of making a mixture comprising tiacumicin B.
However, MCC intended exclusively
for use in non-humans may contain less than 80% of
Tiacumicin B (with respect to the whole antibiotic
substance, by HPLC assay).
[0036]The term "excipient" refers to an inert substance
added to a pharmacological composition to further
facilitate administration of a compound. Examples of
excipients include but are not limited to, calcium
carbonate, calcium phosphate, various sugars and types
of starch, cellulose derivatives, gelatin, vegetable
oils and polyethylene glycols.
[0037]The term "halogen" includes F, Cl, Br and I.
[0038]The term "macrocycles" refers to organic molecules
with large ring structures usually containing over 10
atoms.
[0039] The term "18-membered macrocycles" refers to
organic molecules with ring structures containing 18
atoms.
[0040]The term "membered ring" can embrace any cyclic
structure, including carbocycles and heterocycles as
described above. The term "membered" is meant to denote
the number of skeletal atoms that constitute the ring.
Thus, for example, pyridine, pyran and thiopyran are 6
membered rings and pyrrole, furan, and thiophene are 5
membered rings.
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[0041] The term "MIC" or "minimum inhibitory
concentration" refers to the lowest concentration of an
antibiotic that is needed to inhibit growth of a
bacterial isolate in vitro. A common method for
determining the MIC of an antibiotic is to prepare
several tubes containing serial dilutions of the
antibiotic, that are then inoculated with the bacterial
isolate of interest. Following incubation at appropriate
atmosphere and temperature, the MIC of an antibiotic can
be determined from the tube with the lowest
concentration that shows no turbidity (no growth).
[0042]The term "MIC50" refers to the lowest concentration
of antibiotic required to inhibit the growth of 50% of
the bacterial strains tested within a given bacterial
species.
[0043]The term 'MIC90" refers to the lowest concentration
of antibiotic required to inhibit the growth of 90% of
the bacterial strains tested within a given bacterial
species.
[0044] The term "patient" refers to a human or animal in
need of medical treatment. For the purposes of this
invention, human patients are typically institutionalized
in a primary medical care facility such as a hospital or
nursing home. However, treatment of a disease associated
with the use of antibiotics or cancer chemotherapies or
antiviral therapies can occur on an outpatient basis,
=
upon discharge from a primary care facility, or can be
prescribed by a physician for home-care, not in
association with a primary medical care facility.
Animals in need of medical treatment are typically in
the care of a veterinarian.
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[0045]The term "pharmaceutically acceptable carrier"
refers to a carrier or diluent that is pharmaceutically
acceptable.
[0046]The term "pharmaceutically acceptable salts" refers
to those derived from pharmaceutically acceptable
inorganic and organic bases. Salts derived from
appropriate bases include alkali metal (e.g., sodium or
potasium), alkaline earth metal (e.g., magnesium),
ammonium and N(C1-C4 alky1)4+ salts, and the like.
Illustrative examples of some of these include sodium
hydroxide, potassium hydroxide, choline hydroxide,
sodium carbonate, and the like.
[0047]The term "pharmaceutical composition" refers to a
mixture of one or more of the Tiacumicins described
herein, or physiologically acceptable salts thereof,
with other chemical components, such as physiologically
acceptable carriers and/or excipients. The purpose of a
pharmaceutical composition is to facilitate
administration of a compound to an organism.
[0048]The term "physiologically acceptable carrier"
refers to a carrier or diluent that does not cause
significant irritation to an organism and does not
abrogate the biological activity and properties of the
administered compound.
[0049]The term "pseudomembranous colitis" or "enteritis"
refers to the formation of pseudomembranous material
(i.e., material composed of fibrin, mucous, necrotic
epithelial cells and leukocytes) due to inflammation of
the mucous membrane of both the small and large
intestine.
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[0050]The term "Tiacumicin" as used herein refers to a
family of compounds all of which comprise the 18-
membered macrocycle shown below in Formula I:
H3C =
H3C OH
V
CH3
H3C
1,10 R2
H3CI9R3
Formula I
[0051]The term "Tiacumicin B" as used herein refers to
the 18-membered macrocycle shown below in Formula II:
OH
/ ss
OH
R
0 OH
CI
12
000e,x0 mg 41
1 0 OH
0 0
0 HO 0
19
OH
Formula II (RT ratio 1.0)
[0052]The term lipiarmycin A4 as used herein refers to
the 18-membered macrocycle shown below in Formula XIV:
OH
HO
0 /
0 I 0 CH3
CI
o o o
OH Me0 OH HO OH
[0053] In accordance with one embodiment of the present
invention, after multiple dose oral administrations, low
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MCC levels were detected in plasma, most of which fell
below the limit of quantification. By contrast, fecal
levels in both studies were extremely high, exceeding
10,000 times the MICH (0.125 pg/mL) versus C. difficile.
[0054]In accordance with one embodiment of the present
invention, recurrence of C. difficile -associated
diarrhea can be inhibited in a patient by administering
MCC in an amount and for a duration effective to inhibit
recurrence of C. difficile but with a lack of effect on
normal gut flora in the patient.
[00155]In *accordance with one embodiment, the daily oral
dosage of MCC for CDAD will range from between about 50
mg to about 1.0 grams of active agent per day,
preferably, from between about 100 mg to about 600
milligrams per day. Generally, treatment will be
continued for a time period ranging from between about 3
to about 15 days. Greater or lesser amounts of drug and
treatment intervals may be utilized as required. For
example, according to the results of a clinical study
hereinafter reported, a dosage of about 100-400
milligrams of MCC per day, over the course of from about
ten days, proved effective in treating CDAD with minimal
clinical recurrence.
[0056]In accordance with one embodiment of the present
invention, the mixture can be made by the following
general process.
[0057]MCC-producing bacteria was grown in vessels ranging
from shake flasks to large "batch" fermenters. For
producing substantial quantities of MCC, submerged
aerobic fermentation in tanks is utilized. However,
small amounts may be obtained by shake-flask culture.
For tank fermentation, it is preferable to use a
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vegetative inoculum. The vegetative inoculum is prepared
by inoculating a small volume of culture medium with the
spore form, mycelial fragments, or a lyophilized pellet
of the organism to obtain a fresh, actively growing
culture of the organism. The
vegetative inoculum is
then transferred to a larger tank where, after a
suitable incubation time, the MCC antibiotic is produced
in much improved yield. It may be
necessary to add
small amounts of an antifoam agent to large-scale
fermentation media if foaming becomes a problem.
[0058]The production proceeds in a control medium with
other additives/ingredients to improve the production.
A liquid-submerged, stirred-culture process is used for
the production of MCC. Fermentation is carried out at a
temperature range of 25 00 to 37 C. The consumption of
the carbon source is carefully monitored and an
additional amount of carbon source is added as needed.
The pH of the fermentation is preferably maintained
between about 6.0 to about 8Ø MCC is
produced and
accumulated between 3 to 15 days after inoculation of
the fermentation.
[0059]Commercially available adsorbent resins were found
to enhance the yield and recovery efficiency of MCC
during the fermentation. Adsorbents are preferably
present in the range between 0.5-15% by weight. MCC was
recovered in exceptional yield (> 100 mg/L broth) from
the fermentation broth by resin absorption and eluted
from the resin and mycelium by washing with solvents of
various polaritie;?
[0060]MCC was first captured from the broth during
fermentation using adsorbent resins such as Amberlite
resin (XAD-16). Upon
completion of fermentation, the
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solid mass (including the adsorbent resin) is separated
from the broth by sieving. The solid
mass are eluted
with ethyl acetate then concentrated under reduced
pressure.
[0061]Upon completion of fermentation, the solid mass
(including the adsorbent resin) is separated from the
broth by sieving. MCC is
eluted from the resin with
organic solvents such as ethyl acetate, methanol,
acetonitrile or a mixture of two or more organic
solvents. The
extract is then concentrated under
reduced pressure. This residue is further purified by
trituration with low polarity solvents such as hexanes,
heptanes, methylcyclohexane, or by partitioning between
two phase solvent systems such as: ethyl acetate/water;
ethyl acetate/aqueous sodium chloride solution;
methanol/hexane, acetonitrile/hexane or other mixtures
of two or more solvents in various ratios and
combinations or by column chromatography eluting with an
appropriate organic solvent system. The
current
purification process of MCC is based on medium-pressure
reverse-phase (0-18) column using 50:50:1 CH3CN/1-120/AcOH
or 70:30:1, Me0H/H20/AcOH as eluent. The
fractions
contain desired MCC were washed with brine and the
concentrated. The
residue was dissolved in ethyl
acetate and washed with water and organic layer was
evaporated to dryness to provide a pale yellow foam
which was again washed with isopropyl alcohol and dried
under reduced pressure to yield a white powder. Combine
fractions having purity >88%. Concentrate fractions to
one-half of original volume. Filter
precipitate and
wash filter cake with water. The solid was dried under
high vaccum overnight to give a white powder and
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analyzed by HPLC. Typically, the mixture comprising
tiacumicin B as major components ranged from 90% to 99%,
lipiarmycin A4 (0.1 % to 5%), and at least one or more
of the macrocycles of formula III-XIV described above.
EXAMPLES
[0062] The following examples are provided by way of
describing specific embodiments of the present invention
without intending to limit the scope of the invention in
any way.
[0063] The mixture used in the following examples is
prepared in accordance with the process of making
described above. The following table shows composition
of several exemplary mixtures made in accordance with
the present invention.
API mixture
'profile
fold
wild wild wild wild wild wild
increase
strain type type type type type wild type type mutant mutant
Compound d in MIC
n/a
relative
Trial lA 1B 19 1B 2A 2A 2A (crude) 2B to
B-
9216100 9216190 921619 9216190 9316100 9316100 066004
tiacumic
Lot # 1 1 02 3 1 93161901 2 F7502 1 n B
RRT % % % % %
0.32 - 0.06 0.07 - - 0.06 - - 0.06
0.39 - - 0.01 - - _ _ _ 0.02
0.49 - 0.19 0.13 - - - 0.15 - 0.05
0.71 - - - - - - 3.23 0.02 09-1416 2-4x
0.75 0.46 0.26 0.16 0.17 0.32 0.08 0.12 0.49 0.35
0.79 0.11 0.09 0.07 0.06 0.05 0.08 - 0.72 0.21
=
0.81 0.08 - - - - - - 1.01 0.08 09-1415 8-16x
0.84 0.05 0.04 0.18 0.12 - 0.87 - 3.96 0.25 09-1417 2-4x
0.86 - _ _ _ - - _ -
0.88 - _ - - - - - 3.20 -
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OP-1405
(lipiarmyci
0.89 0.24 0.37 0.61 0.32 0.69 0.50 4.85 1.13
n A4) 1-4x
OP-1431
(tiacumicin
0.92 0.44 0.51 0.42 0.21 0.19 0.16 . 0.17 1.74 0.15 F) 8x
OP-1432
(tiacumicin
0.95 - - - - - - 0.08 0.63 0.06 C) 8-16x
0.96 0.27 0.44 0.65 0.35 - - 0.15 - -
0.98 0.25, - - - - - - -
OP-1441
(tiacumicin
B)
1.00 95.54 97.26 96.40 98.78 98.16 98.55 98.36 73.61 95.6 lx
1.03 - - - - - - 0.22 - 0.22
1.04 0.29 - - - - - - -
1.05 0.37 0.24 0.60 - - - - - -
1.07 0.43 - 0.16 - 0.08 - - - 0.09
OP-1434
(tiacumicin
1.10 0.90 0.31 0.36 - 0.26 0.10 0.10 0.37 -
A) >32x
1.11 - - - - - - - - 0.81
1.13 - - - - - - - - 0.55
1.13 0.32 0.20 0.13 - 0.24 0.11 0.14 1.64 -
09-1435 2x
1.14 0.19 - - - - - - - -
1.19 0.04 - - - - - - 1.50 - 09-1437 2x
1.23 - - - - - - 2.61 0.30 09-1402 2-4x
[0064]The HPLC assay is conducted in accordance with the
following procedure.
[0065] Mobile Phase A: Add 2.0 mli of trifluoroacetic
acid to 2L of HPLC water, filter and degas.
Mobile Phase B: Add 1.0 mL of trifluoroacetic acid
to 2L of Acetonitrile, filter and degas.
Column: 4.6X150 mm column that contains octyl
silane chemically bonded to porous silica or ceramic
micro-particles 3 to 10 pm in diameter (e.g., Zorbax
Eclipse XDB-C8, 3.5 pm).
Detector: 230 nm.
Flow rate: About 1.0 mL/min.
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Injection volume: About 10 }IL.
Run time: About 10 pL.
Diluent: 100% acetonitrile.
Gradient program: Time (Min) %Mobile Phase A
Mobile Phase B
0 60 40
3.0 50 50
14.0 39 61
14.5 60 40
Note: Retention time of the mixture must be within 8-12
minutes.
Standard Preparation: Accurately weigh about 20 mg
of the mixture into a 100 mL volumetric flask, dissolve
in and dilute to volume with Diluent.
Sample Preparation: Accurately weight about 20 mg
of the mixture in a 100 mL volumetric flask. Add about
60 mL Diluent and vortex to dissolve. Dilute to volume
with Diluent and mix.
System Suitability: Chromatography the Standard
preparation and record the peak responses as directed
under Procedure. The relative standard deviation of
tiacumicin B peak areas for five replicate injections is
NMT 2.0%, the tailing factor of tiacumicin B areas is
NMT 2Ø
Procedure: Inject about 10 pL of Diluent.
Separately inject equal volumes (about 10 pL) of
Standard and Sample preparations, record the
chromatograms and measure the detector responses for
major peaks.
Relative Retention Time:
Related Substance RT ratio
Compound of formula II (tiacumicin B) 1.0
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Compound of formula III 0.71
Compound of formula IV 0.81
Compound of formula V 0.84
Compound of formula VI (tiacumicin F) 0.92
Compound of formula VII (Tiacumicin C) 0.95
Compound of formula VIII (Tiacumicin A) 1.10
Compound of formula IX 1.13
Compound of formula X 1.19
Compound of formula XI 1.24
Compound of formula XII 1.39
Compound of formula XIII 1.48
Compound of formula XIV (Lipiarmycin A4) 0.89
Calculations: Calculate the assay value using the
following formula:
Wsd _______________ (mg) SmpDfr __ (m1)
Assay,54= x x Px -x100
SeciD11(0.1) Wartp (ing) x
Where: Ru-tiacumicin B peak area obtained from the
assay preparation.
Rs-tiacumicin B peak area obtained from the
Standard preparation.
P=Purity of Reference standard, including
water factor.
Wstd-Standard weight (mg)
StdDi1=Standard dilution (mL) .
Wsmp--=Sample weight (mg) .
WF=Sample water factor.
Discard peaks originated fro Diluent and calculate
the percentage w/w of individual and total related
substances by the formulae:
individualrelutedsubstancorM win) 1-31 xpp x 100
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Where:Ri-Related substances peak area obtained from
the Sample Preparation.
Rii=tiacumicin B peak area obtained from the
Sample Preparation.
RFir--Related Substance response factor (RF1=1.0
for all related substances.)
[0066]In addition, a typical HPLC profile of the mixture
in accordance with the present invention is shown in
Figure 5. The compounds contained in the mixture, e.g.,
compounds of formula II-XIV, may be found in the HPLC
profile based on their RT ratio. Par-101 in
Figure 5
represents tiacumicin B with RT ratio being 1Ø
P067]The above mixture (50 mg) is then mixed with 100 mg
Avicel PH 102, FMC (microcrystalline cellulose) in a
size 1 capsule shell.
Example 1. Effect of Inoculum, pH, and Cations on the In
Vitro Activity of MCC Vs. Clostridium difficile
[0068]The MIC values measured for many antibiotics are
known to be affected by environmental variables such as
pH, the concentration of divalent cations such as
calcium and magnesium, and the bacterial density. The
dependence of the antibacterial activity on these
factors is an important consideration, particularly for
an antibiotic that targets bacteria in the gut, where
these parameters can vary greatly with the diet and
disease state. m
[0069]The sensitivity of the MIC to these environmental
variables may also be an important factor to consider
when designing methodology for future in vitro testing.
The Clinical and Laboratory Standards Institute, CLSI
(formerly NCCLS) recommends using Brucella agar
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supplemented with vitamin Kl and hemin for Minimal
Inhibitory Concentration (MIC) determination for
anaerobes. The level of divalent cations in this medium,
however, is not standardized. Moreover, the pH of the
media used under anaerobic glove box may also vary under
different gas mixtures. Anaerobes are typically
incubated in a mixture of nitrogen, hydrogen, and carbon
dioxide, and the presence of CO2 will acidify the medium
and can be a significant source of variability. The
inoculum size may also be difficult to standardize given
the variety of atmospheric conditions available for
anaerobic susceptibility testing (H2/CO2 generator,
evacuation/replacement method, or anaerobic chamber).
The anaerobic conditions available to each lab will
determine the duration of organism exposure to aerobic
atmosphere during bench top manipulations and anaerobic
equilibration, and thus affect culture viability and
experimental result.
[0070] In this study, we examined the effect on MIC of the
level of the divalent cations calcium and magnesium, pH
(from 5 - 8), inoculum density (over 3 orders of
magnitude), and also the variability from lot to lot of
Brucella broth .
[0071]Materials and Methods
Bacterial strains:
[0072] Laboratorystrains of Clostridium difficile 9689,
700057, 43255, 17857 and Eubacterium lentum 43055 were
obtained from American Type Culture Collection (ATCC).
All strains were streaked onto brucella agar plates,
supplemented with hemin, and vitamin K from frozen
stocks maintained at - at 78 C in 10% glycerol prior to
use.
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MIC Testing:
[0073]Current CLSI procedures (4) for anaerobic broth and
agar dilution were used for MIC evaluation. Broth
dilution is not a validated method for MIC testing of
Clostridium; however, due to potential inaccuracy of
measuring the pH of solid agar after equilibration
inside the anaerobic chamber, both methods were used and
compared for the assessment of pH effects.
Inoculum density effect on MIC values:
[0074]The effects of inoculum density on susceptibility
of C. difficile to MCC and vancomycin were determined
using the agar dilution method (4). The inocula were
prepared by first making a suspension of -108 cfu/mL and
then serially diluting the suspension by 10-fold factors
to obtain a culture density range between 105 - 108
cfu/mL, to give spot densities of 102 - 105 cfu/spot.
pH effect on MIC values:
[0075]The susceptibility of C. difficile to MCC was
evaluated over a pH range of 6 - 8 using both agar
dilution and microbroth dilution methods.
[0076]Using the agar dilution method, the MIC of MCC was
determined over a pH range of 6.2 - 8.0 against C.
difficile strains in two separate experiments. In order
to achieve the desired anaerobic pH for susceptibility
testing, buffer (100 mM of NaH2PO4 or TAPS [N-
Tris(hydroxymethyl)methy1-3-aminopropanesulfonic Acid])
was added to media at pH 7 and 8, respectively. Even
with strong buffering, the pH shifted slightly following
equilibration in the anaerobic gas, and thus in some
cases media was titered in ambient air to above the
desired anaerobic pH. The actual pH was always confirmed
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following equilibration inside the anaerobic chamber.
Vancomycin, used as a control, was tested only at pH 7.
[0077]Using the broth microdilution method, the MIC
values of MCC and vancomycin were determined over a pH
range of 6 - 8 against C. difficile strains in 3
separate series. In the first series, unbuffered
Brucella broth was titrated in ambient air to obtain a
pH range from 5 - 9. However, anaerobic equilibration of
media in the glove box environment (10% H2 / 5% 002 / 85%
N2) lowered the pH of the media, resulting in an
anaerobic pH range from 5 - 7.5 (as tested using a
portable pH meter with a flat-bottomed pH probe
calibrated with buffer standards outside the glove box,
then transferred inside). For subsequent experiments,
buffer was added to media to resist pH shifts caused by
anaerobic equilibration. In the second series, 10 mM
buffer [NaH2PO4 = H20 pH 7.0, MOPS pH 8.0, or TAPS pH
9.0, pH values in ambient air) was added to media with
pH values greater than 6 to obtain a pH range from 6 -
7.6 after anaerobic equilibration. In the third series,
the buffer concentration was increased to 100 mM for pH
treatments above 6 to obtain an anaerobic pH range from
6 - 8.1.
Divalent cation concentration effect on MIC values:
[00781 The agar dilution method was used to determine the
effect of calcium and magnesium ion concentrations on
susceptibility of C. difficile strains to MCC. The level
of divalent cations in the Brucella broth as acquired
from the manufacturer were determined by the Laboratory
Specialists, Inc. Additional amounts of divalent cations
were added (in the form of calcium or magnesium
chloride) in order to give calcium ion concentrations of
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2.1, 3.0 and 5.7 mg/dL and magnesium ion concentrations
of 3.3, 4.5, and 7.5 mg/dL.
Reproducibility of MCC MIC values with different
commercial lots of media:
[0079] Using the CLSI agar dilution method, susceptibility
of C. difficile to MCC was also examined with three
different commercial lots of Brucella agar, from BBL
(lot #30768960, 211086, and 3167036), supplemented with
different lots of vitamin K (Sigma lot # V-3501 and
0214010) and hemin (Sigma lot # 072K1221 and 034K7656).
Results
Inoculum density effect on MIC values:
[0080]Tables 1 and 2 demonstrate the effect of inoculum
density on the MIC of MCC and vancomycin against two
strains of C. difficile (ATCC 9689 and ATCC 700057).
Susceptibility of both C. difficile strains to MCC was
unaffected by inoculum concentration from 108 - 108
cfu/ml (102 - 108 CFU/spot), as shown by identical MIC
values obtained for all inoculum concentrations tested.
The MIC of vancomycin, however, increased progressively
with increasing inoculum concentration, with the highest
inoculum density showing a fourfold increase in MIC over
the lowest inoculum density. These results demonstrate
that inoculum density is not a significant factor
affecting the outcome of MCC susceptibility testing of
C. difficile.
Table 1. In vitro activity of MCC (ug/mL) vs. different
inoculum densities of C. difficile ATCC 9689 (102-108
CFU/spot).
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Inoculum
Density ATCC 9689
(cfu/ml) CFU/spot MCC vanc
1.92 x 108 1.92 x 105 0.063 2
1.92 x 107 1.92 x 104 0.063 1
1.92 x 106 1.92 x 103 0.063 1 10
1..92 x 105 1.92 x 102 0.063 0.5
Table 2. In vitro activity of MCC (pg/mL) vs. different
inoculum densities of C. difficile ATCC 700057 (102-105
CFU/spot).
Inoculum Inoculum
Density Density ATCC 700057
(cfu/ml) (cfu/ml) MCC vanc
1.48 x 108 1.48 x 103 0.125 1, 2 20
1.48 x 107 1.48 x 104 0.125 1
1.48 x 106 1.48 x 103 0.125 1
1.48 x 105 1.48 x 102 0.125 0.5
pH effect on MIC values:
N0811Table 3 depicts the effect of various pH values on
susceptibility of C. difficile to MCC as measured by
agar dilution method on two separate days. During the
first run, the highest pH treatment (pH 7.9) showed an
8-fold increase in MIC values over the lower pH
treatments (pH 6.2 & pH 7.2) for both strains of C.
difficile. When a confirmatory run was repeated at the
highest pH (pH 8.0), the MIC value remained high for
both strains. No increase in MCC MIC was observed
between pH 6.2 and pH 7 for either strain.
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[0082]The increase in MIC values with pH did not
consistently correlate with increased growth, thus the
effect of pH on MIC did not appear to be merely due to
the enhanced viability of the organism at higher pH. The
pH 7 treatment had less dense organism spot growth
relative to the pH 6.2 and pH 7.9 treatments.
Table 3. pH effects on agar dilution MIC values
(buffered medium)
Anaerobic pH
6.2 7 7.9 8.0
100 mM NaH,PO4 100 mM TAPS 100 mM TAPS
Organism Drug Unbuffered
pH 7.2 (Air) pH 9.2 (Air) pH 9.2 (Air)
MCC 0.063 0.063 0.5 1
ATCC 9689 1 (pH
Vancomycim 6.7) 4
MCC 0.125 0.125 1 2
ATCC 700057 (pH
Vancomycin 6.7) 4
[0083)Table 4, 5 and 6 represents MIC data from the broth
microdilution susceptibility method performed on three
separate days with pH ranges from 5 to 8.1. In the first
series, in which the medium was unbuffered, the MIC of
MCC at pH 7.5 was 8x greater than the MIC at pH 5.9 for
both C. difficile strains (Table 4). The MIC at pH 5
could not be determined, because the organism failed to
grow at this pH. The buffered (10 mM) pH 7.6 treatment
showed 8-fold and 16-fold increases in MCC MIC over the
pH 6 treatment for C. difficile ATCC 9689 & ATCC 700057,
respectively (Table 5). In the third, strongly buffered
(100 mM) series, similar results were seen with the
highest pH treatment (pH 8.1) showing a 16-fold increase
in MIC over the lowest pH treatment (pH 6) for both
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organisms (Table 6). Vancomycin showed a similar trend
with the highest pH treatment producing MICs 4 - 8 fold
greater than the lowest pH treatment in all three
experiments.
Table 4. pH effects on MIC using unbuffered media
Anaerobic pH (unbuffered)
Organism Drug 5 5.9 6.6 7.1 7.5
ATCC 9689 MCC 0.016 Ø016 0.063 0.125
MCC 0.031 0.063 0.125
vanc = 0.5, 1 0.5, 1 2 4
ty)
vanc o 1 0.5, 1 2 4
ATCC 700057MCC 0.031 0.063 0.125 0.25
MCC .L1 = 0.031 0.063 0.125 0.25
vanc = 0.5 1 1 2, 4
vanc 0 0.5 1 1, 2 2, 4
Table 5. pH effects on MIC using weakly buffered media
(10 mM)
Anaerobic pH
6 6.7 7.2 7.6
10 mM Na8l,PO4 10 mM MOPS 10 mM TAPS
Organism Drug pH 6.0 (Air) pH 7.0 (air) pH 8.0 (Air) pH 9.0 (Air)
ATCC 9689 MCC -0.016 0.031 0.063 0.125
MCC -.<Ø016 0.031 0.063 0.125
vanc 0.5 1 2 4
vanc 0.5 1 2 4
ATCC 700057 MCC 0.031 0.063 0.125 0.5
MCC 0.031 0.063 0.25 0.5
vanc 0.5 1 2 4
vanc 0.5 1 2 4
Table 6. pH effects on MIC using strongly buffered
media (100 mM)
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Anaerobic pH
6 6,8 7.5 8 8.1
100mM NaHg0, 100mM MOPS 100 uM TAPS 100 mM
TAPS
Organism Drug pH 6.0 (Air) pH 7.0 (Air) pH 8.0 (Air) pH
9.0 (Air) pH 9.5 (Air)
ATCC 9689 MCC -Ø016, 0.0310.031 0.125 0.25, 0.50.25, 0.5
MCC 0.031 0.125 0.25 0.25
vanc 1 1 4 >8 >8
vanc 0.5 1, 2 4 >8 >8
ATCC 700057 MCC 0.031, 0.063 0.063, 0.1250.25 1 0.5
MCC 0.031 0.063, 0.1250.25 0.5, 1 0.5
vanc 1 2 4 8 8
vanc 1 2 4 8 8
[0084]Assay plates at all pH treatments were also
visually examined for overall growth. In the first
series, which utilized unbuffered broth, overall culture
turbidity increased with increasing pH. The same trend
was observed in the second series, which utilized 10 mM
buffered broth, except the culture turbidity was the
same for pH 7.2 and pH 7.6. In the third series, culture
turbidity was more equivalent across the pH treatments,
with the exception of pH 7.5, which was the most turbid.
[0085] Overall, with both methods of susceptibility
testing and across varying concentrations of buffer
salts, the MIC values of MCC and vancomycin increased
with increasing pH for both strains of C. difficile.
Divalent cation concentration effect on MIC values:
[0085]Measurement of the calcium and magnesium levels in
commercial Brucella broth showed calcium and magnesium
ion concentration of 21 and 33 mg/L, respectively.
Various additional amounts of divalent cations were
added, and MCC MIC values for C. difficile strains were
tested at three different concentrations of calcium ions
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(21, 30 and 57 mg/L) and three different concentrations
of magnesium ions (33, 45 and 75 mg/L). The MIC values
remained the same in all types of media. C. difficile
9689 had MIC value of 0.063 jig/m1 and C. difficile
700057 with MIC value of 0.125 jig/m1 in media with
varying concentrations of cations. Vancomycin, which was
tested as a control with supplemented Brucella agar
without any extra calcium or magnesium as control during
the experiments, demonstrated the expected MIC value of
1 jig/ml for all runs (Tables 7 and 8).
Table 7. In vitro activity of MCC in supplemented
Brucella agar with different divalent cation
concentrations
Calcium C. difficile C. difficile
concent in(ATCC 700057) (ANCC 9689)
Drug Brucella agar 20
media (mg/L)
MIC ( g/mL) MIC ( g/mL)
33 0.125 0.063
45 0.125 0.063
MCC 75 0.125 0.063 25
Vancomycin 33 1 1
Table 8. In vitro activity of MCC in supplemented
Brucella agar with different divalent cation
30 concentrations
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Magnesium C. difficile C. difficiA
concent in(ATCC 700057) (AMCC 9689)
Drug Brucella agar
media (mg/L)
MIC ( g/mL) MIC ( g/mL)
21 0.125 0.063
30 0.125 0.063
MCC 57 0.125 0.063
Vancomycin 21 1 1
MCC MIC values with different commercial lots of media:
[0087]Three different lots of supplemented Brucella agar
media were used on three separate days to compare the
activity of MCC against C. difficile strains. The MIC
assays were controlled by testing the activity of the QC
organism, Eubacterium lentum vs. clindamycin which was
within the CLSI (NCCLS) acceptable ranges, i.e. 0.06 -
0.25 g/mL. Another control step for the MIC assays was
to include metronidazole and monitor its activity vs.
C . difficile strains, which in our laboratory has been
shown to have MIC values ranging between 0.25 - 0.5
g/mL. As shown in Table 9, the activity of MCC vs. C.
difficile was not affected by different lots of
supplemeted Brucella agar. All controls demonstrated
activities within established ranges.
Table 9. In vitro activity of MCC tested with three
different lots of media
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MIC values (ug/m1)
Bacteria Metronidazole Clindamycin MCC
(AMC 0 Day 1 Day2 Day 3 Day 1 Day2 Day3 Day 1 Day 2 Day 3
C. difficile
(9689) 0.5 0.5 0.5 4 4 4 0.25 0.25 0.125
C. difficile
(43255) 0.5 0.5 0.5 8 4, 8 8 0.25 0.5 0.25
C. difficile 0.125,
(17857) 0.25 0.5 0.5 4 2 4 0.25 0.25 0.125
Eubacterium
lentum 0.125, 0.06,
(43055) 1 0.25 1 0.25 0.25 0.125 0.25 0.25
0.125
Conclusions
[0088]In contrast to vancomycin, the activity of MCC vs.
C. difficile was unaffected by inoculum concentrations,
in the range of 102 - 105 cfu/spot.
[0089]The susceptibility of C. difficile to MCC was
unaffected by cation concentrations (calcium ion in the
range of 2.1 - 5.7 mg/dL and magnesium concentration of
3.3 - 7.5 mg/dL), and by various commercial lots of
media.
[0090] The MIC values for both MCC and vancomycin
increased with increasing pH over a pH range of 6 - 8.
The high MIC values at basic pH may be due to
deprotonated form of the phenolic hydroxyl groups of
both compounds above their pKa, where they form a
charged species that is expected to be less permeable to
bacterial cells. In contrast, below the pKa (7.22 for
MCC), the antibiotics will be mostly protonated, and
thus should permeate the cell membrane more efficiently.
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[0091] Organism density generally increased with
increasing pH; the dependence of growth density, but not
MIC, on pH was reduced in the presence of buffering
agents. Though organism density was positively
correlated with basicity in the absence of buffer, it is
unlikely that MIC trends are the result of the effect of
pH on organism density alone. This is because the same
relationship between MIC and pH was observed in buffered
experiments where organism density was more equivalent
across pH treatments, presumably due to the differential
effect of buffer type on organism growth.
Example 2. Safety, Pharmacokinetics and Outcomes of MCC
in Healthy Subjects and Patients with Clostridium
diffiche-Associated Diarrhea (CDAD)
Phase 1B-MD.
[0092]Synopsis. This was an oral, multiple-dose, double-
blind, randomized, placebo-controlled, dose escalation
study conducted at the University of Miami Division of
Clinical Pharmacology, Miami, Florida. Richard Preston,
M.D. served as the Principal Investigator for this
trial. The tolerability and pharmacokinetics of multiple
oral doses of MCC were evaluated in a total of
24 healthy volunteer subjects. The oral doses of MCC
evaluated (in 3 groups of 8 subjects each, with 6 active
and 2 placebo) were 150, 300, and 450 mg (in powder-
filled capsules containing 50 mg of study drug)
administered daily after a morning breakfast for
10 consecutive days. Subjects were dosed and monitored
on a combined inpatient/outpatient basis. Subjects were
admitted to the research unit on Day 0 and again on Day
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9 of the 10-day dosing period, and stayed for up to 48
hours after each admission. Subjects were discharged on
Day 2 and Day 11 after completing the scheduled events
and procedures. During the outpatient period, subjects
reported daily to the research unit for dosing and
stayed for 3 hours under observation.
[0093]Serial blood, urine, and fecal samples were
collected at various time points/intervals during the
multiple dosing periods. Plasma, urine, and fecal
concentrations of MCC were determined for
pharmacokinetic analysis. A follow-up examination was
scheduled on Day 17 of each study period before subjects
exited from the study. Study subjects were closely
monitored for the occurrence of any adverse experiences
or abnormal laboratory test findings throughout the
treatment periods and at the study follow-up. See,
Figure 1, Phase 1B-MD Dosing schedule.
Phase 2A.
[0O94} Synopsis. This was a dose-finding study to select a
safe and effective dose of MCC. Subjects were randomized
to receive either 100 (50 mg every 12 hours), 200
(100 mg every 12 hours), or 400 (200 mg every 12 hours)
mg/day for 10 days followed by clinical evaluation.
Subjects recorded all symptoms on daily diary cards.
Particular attention was to be given to stool frequency
and consistency, the presence of blood in the stool, and
abdominal discomfort. Laboratory assessments were
performed at Screening for entry. and at End of Treatment
(Day 10-12) or withdrawal
(whichever was sooner).
Clinical observation and diary card evaluation were
performed at End of Treatment (Day 10-12). Patient
interviews were conducted on treatment Day 2 through 9,
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Day 17, and Day 52. For entry inclusion criteria, assay
for Clostridium difficile toxin was performed. For
subjects that failed to respond to MCC treatment, and in
the event of clinical recurrence, both C. difficile
toxin assay and culture were performed. Clinical,
laboratory, and microbiological assessments were also
performed at exit for subjects that failed to respond to
treatment. Pharmacokinetic plasma samples were taken 0.5
hr prior to dosing and 2 hr after dosing on the first
and last days of dosing.
[0095] Key Inclusion Criteria. Subjects were patients with
C.difficile associated diarrhea as defined by: 1)
diarrhea (a change in bowel habits, with 3 or more
unformed bowel movements in 24 hours, or more than
6 loose or watery stools within 36 hours.) and 2)
presence of either toxin A or B of C. difficile in the
stool.
[0096]Key Exclusion Criteria Subjects could not have 1)
severe or life-threatening CDAD 2) life-threatening or
serious disease unrelated to CDAD, 3) concurrent use of:
vancomycin, metronidazole, bacitracin, or related drugs.
(If the Investigator felt the clinical imperative to
begin treatment before knowing the laboratory result for
stool toxin, up to 24 hours, but no more than 3 doses,
of treatment with metronidazole and/or vancomycin was to
be allowed.); any drugs used for the treatment of CDAD;
or other antibiotics 4) history of ulcerative colitis or
Crohn's disease and multiple recurrences (defined as
more than one recurrence) of CDAD within the past three
months. (Subjects with a single recurrence of CDAD were
permitted to enroll.)
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Schedule of Events
Table 10. Schedule of Evaluation Procedures in the phase 2A
study
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Assessments Day 1 Treatment End of Completion Day 52
Screen/ Days 2 Treatment Day 17 Follow-
Enrollment through 9 or Day 10 up
Informed X
Consent
Inclusion/Ex X
clusion
Medical X
History
Physical X X
Examination
Vital Signs X X
12-Lead ECG X X
Clinical X X
Laboratory
Tests
Stool Sample X X
PK Blood X X
Sampling'
Fecal PK X
Sampling
Adverse X X X
Events
Concomitant X X X X X
Medication
Pregnancy X
Test
Diary Card X X X
Review
Study X X X
Medication
Administrati
on
Subject X X X X X
Interview
'Blood samples for pharmacokinetics taken 0.5 hr prior to and 2 hr
post administration on the first and last days of dosing
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[0097] Endpoints . At the end of therapy, the investigator
determined if the subject had been cured of failed. In
addition, the time to resolution of diarrhea (defined as
resolution to <3 loose or watery stools per day) and the
complete relief of symptoms of CDAD by day 10 of therapy
(complete relief was resolution to total stools
per
day, whether) loose or firm; and absence of fever,
elevated white blood cells, or abdominal pain) were
tracked as primary endpoints, and recurrence within 6
weeks following therapy (recurrence of diarrhea, defined
as 3 or more loose/watery stools per day, with a
positive toxin test) was tracked as a secondary
endpoint.
Analysis
Safety Population:
[0098]The safety population was to include all randomized
subjects who received at least one dose of study
medication and had safety information available.
Efficacy Population:
[0099]Clinical success or failure was determined in
patients treated per protocol. The population analyzed
for time to resolution of diarrhea and complete relief
of symptoms was the modified intent to treat population
(mITT), consisting of all randomized subjects who
received at least one dose of study medication, had a
history of diarrhea, and had 3 or more loose stools in
24 hours and a positive C. difficile toxin at baseline.
[00100] Time to
resolution of diarrhea was defined as
time (in days) from the first dose of study medication
to the resolution of diarrhea; time to resolution of
diarrhea was compared among the three treatment groups.
The cessation day of diarrhea was defined as the first
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day that <3 unformed stools (watery or loose) within a
24 hour period occurred and was sustained for the
duration of treatment up to study Day 10. Resolution of
diarrhea was assessed during a 10 to 12 day period
utilizing the subject diary data.
Complete Relief of symptoms of CDAD:
[00101] Complete relief of symptoms of CDAD was
defined as resolution to bowel
movements per day (as
recorded on the patient diary) without other associated
signs/symptoms such as fever (37.7 C), abdominal pain
(no response on diary) and elevated WBC (normal
laboratory range of WBC) by Day 10 of the study. If any
variable was missing, this outcome was considered
unknown.
Clinical recurrence rate:
[00102] Clinical recurrence was defined as unformed
stools (loose or watery) and a positive stool for
C. difficile toxin A or B within 6 weeks posttreatment.
[00103]
RESULTS
Enrollment and Demographics
[00104] The following sections summarize the
enrollment and demographic characteristics of the study
populations in the phase 15-MD and 2A trials. A total of
24 healthy subjects were enrolled for the phase 1B-MD
study. Alternate male and female subjects were enrolled
to provide an even split between the sexes. Subjects
ranged in age from 38 - 62 years (average 51.6 7.5
yr), in weight from 55.5 -
90 kg (average 71.5 9.2
kg), and in height 147 - 183 cm (average 164.8 10.8
cm.)
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[00105] In the phase
2B study, a total of 49 subjects
were enrolled. One subject withdrew consent and was
dropped from the study prior to receiving any study
drug, and was not evaluable for either safety or
efficacy. One subject (400 mg dosing group) had >6
bowel movements in 36 hours, but <3 bowel movements in
the prior 24 hours, and could not be evaluated for time
to resolution of diarrhea but was evaluable for clinical
response and safety analyses. Three patients were
discontinued after 1 or 2 doses due to removal of
consent (1 subject, 100 mg dosing group), requirement
for additional antibiotics for pneumonia (1 subject, 100
mg dosing group), or inability to take study medication .
(1 subject, 200 mg dosing group). Subject demographics
are listed in Table 11.
[00106] Table 11.
Summary demographics for the Phase 2A study;
demographics for the 48 subjects in the population evaluable for safety are
shown.
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MCC MCC MCC All
100 mg/Day 200 mg/Day 400 mg/Day Subjects
(N=16) (N=16) (N=16) (N=48)
Sex
Female 10 (62.5% 11 (68.8% 9 (56.3% 30
(62.5%
) )
Male 6 (37.5% 5 (31.3% 7 (43.8% 18
(37.5%
) )
Race
Caucasian 14 (87.5% 15
(93.8% 14 (87.5% 43 (89.6%
) )
Black 2 (6.3%) 1 (6.3%)
0 (0.0%) 2 (4.2%)
Asian 0 (0.0%) 0 (0.0%)
1 (6.3%) 1 (2.1%)
Hispanic 0 (0.0%) 0 (0.0%)
0 (0.0%) 0 (0.0%)
Other' 1 (6.3%) 0 (0.0%)
1 (6.3%) 2 (4.2%)
Age(Yrs)
Mean SD 56.3 17.78 53.1 22.97 55.3 17.69 54.8 19.26
Median 54.5 55.5 56.0 56.0
Range 28.0-89.0 18.0-88.0 18.0-90.0 18.0-90.0
Weight (Kg)
Mean SD 69.2114.0 68.4
11.46 67.5 13.5 68.4 12.8
0 6 2
Median 69.3 66.0 65.2 66.0
Range 38.0-89.0 52.0-96.0 40.0-88.2 38.0-
96.0
Height (cm)
Mean SD 163.8 15.52 166.4 9.48 166.2 13.18 165.5 12.8
0
Median 162.1 170.0 163.8 165.0
Range 122.0-187.5 150.0-178.0 142.0-193.0 122.0-
193.0
Calculated Body Mass
Index)'
Mean SD 25.8 3.89 24.9 4.50 24.3 2.52 25.0 3.68
Median 25.0 24.0 24.5 25.0
Range 17.0-34.0 17.0-32.0 20.0-28.0 17.0-34.0
NOTE: values represent number of subjects unless otherwise
indicated.
a Other includes: East Indian, Indian.
b Calculated body mass index is defined as (weight in kg)/(height
in meters)2.
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Efficacy
[00107] In the clinical evaluation of treatment
success or failure at the end of therapy, two patients
in the low dosing group (2/14), 2 patients in the mid
dosing group (2/15), and no patients in the top dosing
group (0/16) were considered treatment failures by the
investigator. Among the subjects (n=41) that were
treatment successes, CDAD recurred in one subject (1/12)
in the 100 mg/day dosing group and one subject (1/16) in
the top dosing group, for a recurrence rate of 2/41 (5%)
overall. Both recurrences occurred approximately 1 month
following the end of therapy.
Table 12. Rates of clinical cure and recurrence in the
population treated per protocol.
MCC MCC MCC
100 mg/Day 200 mg/Day 400 mg/Day
Total 14 100 15 100 16 100
Treatment
12 86 13 87 16 100
success
Treatment
2 14 2 13 0 0
failure
Clinical
1 8.3 0 0 1 6.3
recurrence'
' Recurrence of toxin-posivite diarrhea within 6 weeks post-
treatment, evaluated in patients that were clinical successes.
[00108] The time to resolution of diarrhea was defined
as the time for the patient to resolve to less than 3
unformed stools per day, according to the patient's
diary card. In the mITT population, the median time to
relief was 5.5 days, 3.5 days, and 3.0 days for the MCC
100 mg/day, 200 mg/day and 400 mg/day treatment groups,
respectively. The mean time to resolution of diarrhea in
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days was 6.3 3.66 days in 100 mg/day-treated subjects,
4.8 3.56 days in 200 mg/day-treated subjects, and
3.6 2.03 in 400 mg/day-treated subjects. There was no
statistically significant difference in time to
resolution of diarrhea between the 100 mg/day and
200 mg/day treatment groups, and between the 200 mg/day
and 400 mg/day treatment groups; however the difference
between the 100 mg/day and 400 mg/day treatment groups
approached statistical significance (p=0.0506 Kaplan
Meier estimate and p=0.0503 Kruskal-Wallis test).
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Table 13. Time to Resolution of Diarrhea (mITT
population), defined as time to resolve to <3 unformed
bowel movements per day (according to the patient's
diary card)
MCC MCC MCC
100 mg/Da 200 mg/Da 400 mg/Da
P-Value
16 16 15
N (Resolved Diarrhea) 10 12 14
N (Censored: Did not 4 3 1
resolve)e
N (Censored: Dropped from 2 1 0
= study)
N (Censored: Total) 6 4 1
Median Time (Days)b 5.5 3.5 3.0
P-Valuec 0.1912
MCC 100-MCC 200c 0.2901
MCC 100-MCC 400c 0.0506
MCC 200-51CC 400c 0.6143
a Subjects whose diarrhea was not resolved to <3 loose stools/day by day
b Kaplan-Meier estimates
c P-value obtained from generalized Wilcoxon Test.
[00109]
Complete relief of symptoms of CDAD by the end
of treatment, defined as 3 total
bowel movements per
day (whether formed or unformed, as recorded on the
10 patient's diary card), and no fever, elevated WBC count,
or abdominal pain (according to response on patient
diary card) by the 10th day of the study, is shown in
Table 14. Complete relief was achieved by 37.5% of the
100 mg/day treatment group, 50.0% of the 400 mg/day
treatment group, and 86.7% of the 400 mg/day treatment
group. It is worth noting that most patients that did
not have complete relief by day 10 were nevertheless
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treatment successes, had resolution of symptoms by day
17, and did not require further treatment. Three
patients that dropped from the study (one for removal of
consent, one for the requirement of exclusionary
antibiotics, and one for the inability to take oral
medications) are also listed as having no complete
relief.
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Table 14. Complete Relief of Symptoms of CDAD by end
of therapy in the mITT
population , defined as
resolution to 3 total bowel
movements per day
(formed or unformed, as noted on the patient's diary
card) without other associated signs/symptoms such as
fever, abdominal pain, and elevated WBC by Day 10 of
the study
MCC MCC MCC
100 mg/Day 200 mg/Day 400 mg/Day
Complete Relief 6 (37.5) 8 (50.0) 13 (86.7)
No Complete Relief 9 (56.3) 6 (37.5) 2 (13.3)
Required further 2 (12.5) 2 (12.5) 0 (0)
treatment
Required no 5 (31.3) 3 (18.8) 2 (13.3)
further treatment
Dropped from study 2 (12.5) 1 (6.3) 0 (0)
Unknown 1. (6.3) 2 (12.5) 0 (0.0)
[00110] Only 2 subjects (1 subject in the 100 mg/day
treatment group and 1 subject in the 400 mg/day
treatment group) experienced clinical recurrence.
Safety
[001 1 1] In the phase 1B-MD study, MCC was well
tolerated by all subjects at all doses. Fourteen adverse
events were reported, 7 in the 150 mg group, 2 in the
450 mg group, and 5 in the placebo group. The adverse
events are summarized as follows: headache (2),
dizziness (1), weakness (1), fatigue (1), nasal
congestion (1), difficulty swallowing (1), pharyngitis
(1), conjunctivitis (1), upper respiratory infection
(2), rash (1), and pruritis (1). No subjects receiving
MCC had adverse events considered to be drug-related.
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[001121 In the phase 2A study, as shown in Table 15,
4/16 (25.0%) subjects in the 100 mg/day treatment group,
4/16 (25.0%) subjects in the 200 mg/day treatment group,
and 1/16 (6.3%) subjects in the 400 mg/day treatment
group, reported at least one AE during the study. The
highest frequency of AEs was reported in the infections
and infestations body system in the 100 mg/day treatment
group (3/16; 18.8% subjects). There were 2/16 (12.5%)
subjects who reported vascular disorders in the
100 mg/day treatment group and 2/16 (12.5%) subjects who
reported gastrointestinal disorders in the 200 mg/day
treatment group.
=
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Table 15. Incidence of adverse events in the safety
population of the 2A study, summarized by system
organ class and preferred term
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mcc MCC MCC
100 mg/Day 200 mg/Day 400 mg/Day
System Organ Class (N=16) (N=16) (N=16)
Preferred Term xi (%) n (%) n (%)
Total subjects with adverse events 4 (25.0 4 (25. 1 (6.3)
0)
Cardiac disorders 1 (6.3) 0 (0.0 0 (0.0)
)
Cardiac failure congestive 1 (6.3) 0 (0.0 0 (0.0)
)
Gastrointestinal disorders 0 (0.0) 2 (12. 0 (0.0)
5)
Gastrointestinal haemorrhage 0 (0.0) 1 (6.3 0 (0.0)
)
Pancreatitis chronic 0 (0.0) 1 (6.3 0 (0.0)
)
General disorders and 1 (6.3) 1 (6.3 0 (0.0)
administration site conditions )
Chest pain 1 (6.3) 1 (6.3 0 (0.0)
)
Infections and infestations 3 (18.8 1 (6.3 0 (0.0)
)
Bronchitis 1 (6.3) 0 (0.0 0 (0.0)
)
Infection 1 (6.3) 0 (0.0 0 (0.0)
)
Pneumonia 1 (6.3) 0 (0.0 0 (0.0)
)
Staphylococcal sepsis 0 (0.0) 1 (6.3 0 (0.0)
)
Urinary tract infection 1 (6.3) 0 (0.0 0 (0.0)
)
Injury, poisoning and procedural 0 (0.0) 1 (6.3 0 (0.0)
complications )
Fall 0 (0.0) 1 (6.3 0 (0.0)
)
Metabolism and nutrition disorders 0 (0.0) 0 (0.0 1 (6.3)
)
Fluid overload 0 (0.0) 0 (0.0 1 (6.3)
)
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Musculoskeletal and connective 1 (6.3) 0 (0.0 0
(0.0)
tissue disorders )
Pain in extremity 1 (6.3) 0 (0.0 0 (0.0)
)
Nervous system disorders 0 (0.0) 1 (6.3 0 (0.0)
)
Cerebral haemorrhage 0 (0.0) 1 (6.3 0 (0.0)
)
Renal and urinary disorders 1 (6.3) 0 (0.0 0 (0.0)
)
Nephrolithiasis 1 (6.3) 0 (0.0 0 (0.0)
)
Respiratory, thoracic and 1 (6.3) 0 (0.0 0 (0.0) ,
mediastinal disorders )
Dyspnoea 1 (6.3) 0 (0.0 0 (0.0)
)
Vascular disorders 2 (12.5 0 (0.0 0 (0.0)
)
Hypotension 2 (12.5 0 (0.0 0 (0.0)
NOTE: Percentages are the proportions of subjects within that
category.
[00113] Five subjects were reported as having SAEs
during the study (Table 16). In the 100 mg/day treatment
group, one subject had diarrhea of moderate severity and
another subject had severe exacerbation of congestive
heart failure (CHF). In the 200 mg/day treatment group,
one subject had severe staphylococcal sepsis and a
severe cerebral hemorrhage, another subject had a
gastrointestinal hemorrhage of moderate severity, and a
third subject had chest pain of moderate severity. No
subject in the MCC 400 mg treatment group had an SAE.
All SAEs were considered to be unrelated to study drug.
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Table 16. Incidence of serious adverse events in the safety
population of the 2A study
Total
Durati
on of
Treatmen Therap Adverse Durati Relation
Event Stud on of ship to
Subject Age (Days) (Preferred y AS Seven i Study
Number (Yrs) Sex Race a Term) Dayb (Days) ty
Drug' Outcome
MCC100 mg/day
Recover
ad
Without
Caucasi Modera Not
Sequela
314 34 Male an 10 Diarrhea 33 3 te Related e
Recover
Cardiac ad
With
Failure Not
Sequela
400 52 Male Black 10 Congestive 39 12 Severe Related e
MCC 200 mg/day
Not Yet
=
Tarsal Caucasi Staphylococ Not
Recover
200 85 e an 10 cal Sepsis 10 7 Severe Related edd
Not Yet
Cerebral Not
Recover
Haemorrhage 10 7 Severe Related add
Recover
ad
Gastrointes
Without
Femal Caucasi tinal Modera Not
Sequela
208 71 e an 10 Haemorrhage 15 14 te Related e
Recover
ad
Without
Penal Caucasi Modera Not
Sequela
304 59 e an 11 Chest Pain 23 6 te Related e
a Date of last dose of study medication minus date of first dose of study
medication plus
one.
b Study day is calculated as follows: date of onset minus date of girst date
of study
medication plus one.
' Based on Investigator's assessment.
d Subject died.
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Pharmacokinetics
Plasma Concentration Data
[00114] In the phase 15-MD study, after multiple dose
oral administrations, plasma concentrations of MCC were
mostly below the limit of quantification across the dose
range
[00115] Detectable plasma concentrations were found
only in 12 samples from 6 subjects.
[00116] Of the 12 detectable concentrations, only 2
were significantly above the LLOQ, while others barely
exceeded the LLOQ of 5 ng/mL.
[00117] These two concentrations (11.1 and 48.0 ng/mL)
were observed in Subject 021 on Day 1, Hour 1 and just
prior to the tenth dose on Day 10, respectively.
[00118] It is to be noted that the 150 mg dose
produced no detectable concentrations.
[00119] Due to low MCC plasma levels across the dose
range, there were insufficient plasma data points above
LLOQ for pharmacokinetic analysis.
[00120] In the phase 2A study, after multiple dose
oral administrations, plasma concentrations of MCC were
mostly below the limit of quantification but with a dose
dependent increase in the number of samples, and number
of subjects, with measurable plasma concentrations.
[00121] Detectable plasma concentrations were found in
2/15 (13.3%) subjects in the MCC 100 mg/day treatment
group, 9/16 (56.3%) subjects in the MCC 200 mg/day
treatment group, and 13/17 (76.5%) subjects in the MCC
400 mg/day treatment group.
[00122] Observable MCC concentrations ranged from 9.45
to 12.3 ng/mL in the MCC 100 mg/day treatment group,
5.12 to 93.7 ng/mL in the MCC 200 mg/day treatment
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group, and 5.32 to 84.9 ng/mL in the MCC 400 mg/day
treatment group.
[00123] Of the detectable concentrations of MCC in all
treatment groups, the majority (35/41; 85.4%) were under
21 ng/mL.
[00124] Concentrations of MCC over 50 ng/mL were
observed in only 2 subjects, one each in the 200 mg/day
and 400 mg/day dosing groups.
Urinary Excretion Data of MCC
[00125] Levels of MCC in the urine in the phase 1B-MD
study were all below the limit of quantification (LLOQ =
5 ng/mL).
Fecal Concentration Data of MCC
[00126] Table 17 shows fecal concentrations from the
1B-MD study, normalized to the 150 mg dose; fecal MCC
averaged 916.0 pg/g (138.4-1768.9 }.'g/g)=
Table 17. Fecal concentrations of MCC in the phase 1B-MD
study, normalized to a 150 mg dose.
[MCC]
Subject Dose [MCC]
(normalized)
(mg) (pg/g) (ug/g)
Range 150-450 415.1 - 5306.8138.4 - 1768.9
Mean: 916.0
SD: 450.2
[00127] For the phase 2A study, in the MCC 100 mg/day
treatment group (n=11 samples sufficient), fecal MCC
averaged 255.6 pg/g (range: 81.9-558.3 pg/g) at the end
of treatment. In the MCC 200 mg/day treatment group
(n=9 samples sufficient), fecal MCC averaged 441.7 pg/g
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(range: 11.7-786.7 pg/g). In the MCC 400 mg/day
treatment group (n=13 samples sufficient), fecal MCC
averaged 1433.3 pg/g (range: 389.0-3974.8 pg/g).
[00128]
Table 18. Fecal concentrations of MCC at the end of
treatment in the phase 2A study.
[MCC] [MCC]
Dose N
range average
(mg/day) (Pg/g) (pg/g)
100 11 81.9-558.3 255.6
200 9 11.7-786.7 441.7
400 13 389.0-3974.8 1433.3
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CONCLUSIONS
[00129] In
summary, the present studies show that MCC
is well-tolerated after multiple oral doses up to 450
mg, achieves high levels at the site of action, and
shows promising results in the treatment of C.
difficile-associated diarrhea.
[00130] This study also found 1) there were no
treatment-emergent adverse events felt to be possibly
drug related in either study, 2) after multiple dose
oral administrations, low MCC levels were detected in
plasma, most of which fell below the limit of
quantification. Consequential to low plasma
concentrations, no intact MCC was detected in the
collected urine of the 1B-MD study.3) by contrast, fecal
levels in both studies were extremely high, exceeding
10,000 times the MICH (0.125 pg/mL) versus C. difficile,
4) among 45 subjects treated with a full course of
therapy, only four subjects were considered failures
prior to or at the end of 10 days of therapy, 2 subjects
in the 50-mg ql2hr and 2 subjects in the 100-mg ql2hr
dose groups. No failures (0/16) were noted in the 200-mg
ql2hr dose, 5) recurrence was observed in only 2
subjects following successful treatment. Both recurred
approximately one month after the end of therapy, 6)
although not statistically significant, the median time-
to-cessation of diarrhea, showed a trend which suggested
that higher doses may be more efficacious. Time-to-
cessation of diarrhea was determined to be 5.5 days for
the 50-mg q 12 hr dose group, 3.5 days for the 100-mg q
12 hr dose group, and 3.0 days for the 200-mg q 12 hr
dose group.
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Example 3. MCC is selectively effective against C.
difficile in-vivo, and does not affect major members of
the anaerobic fecal flora: key to a lower relapse rate.
[00131] To test the hypothesis that MCC is selectively
active in-vivo against C. difficile and could be
relatively sparing of the normal anaerobic fecal flora,
quantitative stool cultures were performed on serial
stool samples obtained from patients entered into a
Phase 2A dose ranging clinical trial of MCC (now
designated MCC). Optimal antibiotic therapy of C.
difficile diarrhea should eradicate the vegetative forms
of the pathogen, yet spare major components of the
normal flora presumed to be responsible for colonization
resistance.
Methods
[00132] Patients (n=32) were randomized to receive 50,
100 or 200 mg twice daily of MCC for 10 days. No prior
therapy was given to 24 patients; 8 receive 1 or 2 doses
of standard therapy. As ecologic controls, 7 additional
patients were treated with vancomycin 125 mg qid for 10
days. Fresh stool samples were cultured 10-2'4," for
C.difficile vegetative and spore forms; fecal filtrates
were tested for cytotoxin B by cell assay. At study
entry and day 10, aerobic and anaerobic fecal flora
cultures, diluted 10-3'5'7'9 , were examined for major
floral shifts. Since Bacteroides group organisms are
ubiquitously present and cultivable, this genera was
selected as a indicator of the integrity of the
microbial flora.
[00133] Detailed method shows as the following.
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1) Single center study in Calgary Health Region
catchment area, population -1 million
2) Randomized open label, dose ranging Phase 2A study
comparing 50 mg, 100 mg or 200 mg Q 12 hourly of
MCC for 10 days p. o. as therapy of CDAD.
3) Following completion of the trial recruitment, a
separate ecology control group of patients who
otherwise would be eligible for the trial were
treated with Vancomycin 125 mg QID for 10 days as a
treatment / ecologic control.
4) Mild to moderate CDAD: >3 but < 12 diarrheal
samples / 24 hours at study entry, positive C.
difficile toxin assay, fever < 39 degrees C, WBC <
30,000/mm3, no vomiting, no severe abdominal
discomfort
5) Primary CDAD or first relapse episode only.
6) Treatment naive if possible. The protocol allowed
up to 3 prior doses of standard therapy, but for
this evaluation, a maximum of 2 doses of standard
therapy was allowed. In this study population, 24
patients were treatment naive
7) No concomitant parenteral antibiotic therapy for
any condition.
8) Serial stool samples: in addition to the original
diagnostic sample, a repeat collection of stool > 5
grams (10-30 grams usually) was obtained at study
entry, at day 4, 7, 10, 14, 21, 28 and 42 days
after study entry
9) For this report, results of day 0 and day 10 stools
are compared for changes in C. difficile counts and
in counts of major genera of the normal colonic
flora.
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10) C. difficile quantitative counts and fecal
filtrate concentrations of C. difficile cytotoxin B
by HeLa cell assay were determined with freshly
passed samples as refrigeration is deleterious to
determination of quantitative counts of C.
difficile.
11) Since Bacteroides group organisms are
considered to be uniformly present in subjects and
in high counts, and is likely one of the major
components of the normal flora conferring
`colonization resistance', this group was used as
an index of suppression of the anaerobic fecal
flora. For patients who failed to show return of
the Bacteroides group species at 10 days,
subsequent samples were processed to document time
of return of this group. If samples were not
immediately processed, aliquots were frozen at -80
degrees C with 15% glycerol / Brain Heart Infusion
Broth for subsequent processing.
12) Media and methods for anaerobic flora cultures
are based on the Wadsworth-KTL Anaerobic Manual,
6th ed, 2002. C. difficile counts were determined
by dilution of the sample 10-2,4,6,8 / gram stool
wet weight on CCFA agar. Spore counts were
determined by treating an aliquot of stool with an
equal volume of 100% ethyl alcohol x 1 hour,
centrifuged, washed twice and resuspended for
quantitative counts.
13) Normal flora cultures were quantified by
dilution 10 -3,5,7f 9 using MacConkey, BAP, m-
Enterococcus agar, Lab M anaerobic blood agar, BAP,
BEE, KVLB, PEA agars incubated for 48 hours before
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initial inspection, and further incubated for up to
7 days.
14) For vancomycin ecologic controls, vancomycin
fecal filtrate concentrations were determined in
triplicate by bioassay using a C. perfringens as
the indicator organism.
15) Differences in microbial counts were
determined after logn transformation using wilcoxon
matched.
Results
[00134] At study
entry, mean log 10CFU + SD vegetative
counts of C. difficile (all MCC patients) were 6.8 +
3.6, range 2-10.95; at day 10, with the exception of one
patient receiving 50 mg, all other patients had C.
difficile quantitative counts reduced < 2 logn/gm feces.
Vancomycin was similarly effective. At study entry,
Bacteroides group counts were <3, 3-8, & 8.5-10 log10
CFU/gm in 1/3 each of patients, with normal counts being
>11. Shifts in the Bacteroides group are shown table 19.
Table 19. Mean + SD of logn CFU of Bacteroides group
counts/gm, feces wet weight
MCC, 50 mg MCC, 100 mg MCC, 200 mg Vancomycin
(n=10) (n=8) (n=11) (n=7)
Day 0 6.64 + 6.64 + 7.04 + 7.39
2.82 2.82 2.87 2.67
Day 10 8.23 + 6.30 + 7.34 + 3.62**
2.60 2.53 3.06 1.90
p * 0.11 0.44 0.56 0.03
* wilcoxon matched pairs signed-ranks test, 2 tailed;
** counts <3 logn =2.90
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[00135] The
followingfigures further illustrate the
results from the study.
Conclusions
[00136] Based on quantitative Bacteroides group
counts, patients with C. difficile diarrhea have
variably impaired normal flora at study entry, with
approximately 1/3 in the 3 log10 CFO/gm range, 1/3 in
counts of 4-7 log10 CFU, and the remainder with higher
counts (none in the normal range of 11-12 log10 CFU).
All dosages of MCC appeared to reduce counts of C.
difficile, as did vancomycin. A dose
dependent
reduction in Bacteroides counts with increasing dosages
of MCC was not observed.
Vancomycin severely impairs .
Bacteroides counts during therapy and although most
patients recover their counts, a minority have prolonged
absence.
[00137]
Based on these data and clinical outcomes
showing a high response rate accompanied by a low
relapse rate, it would appear that the 200 mg dose of
MCC would be an appropriate dosage to undergo further
clinical investigation.
[00138] It will be understood that the scope of the claims
should
not be limited by the preferred embodiments set forth in the examples,
but should be given the broadest interpretation consistent with the
description as a whole.
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