Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF REDUCING THE SEVERITY OF ORAL AND
GASTROINTESTINAL MUCOSITIS
Backp-round of the Invention
The invention relates to methods for reducing the severity of oral and
gastrointestinal mucositis.
Mucositis is a condition characterized by swelling, irritation, and discomfort
of
mucosal linings such as those of the gastrointestinal tract and the oral and
oralpharyngeal cavities, and can result in mouth and throat sores, diarrhea,
abdominal
cramping and tenderness, and rectal ulcerations. This condition occurs in
approximately half of all cancer patients, and is a common side effect of
cancer
treatments involving radiation and/or chemotherapy. The goal of these
approaches to
cancer treatment is to kill rapidly dividing cancer cells but, unfortunately,
other
rapidly dividing cells are killed by the treatment as well, including cells
that line
regions such as the gastrointestinal tract, leading to mucositis. Symptoms of
mucositis generally occur five to ten days after the start of cancer
treatment, and can
take two to four weeks after cessation of treatment to clear. The incidence of
mucositis, as well as its severity, depends on factors such as the type and
duration of
the cancer treatment. Mucositis occurs, for example, in virtually all patients
who are
treated by irradiation of the head and neck. It is also highly prevalent in
patients
treated with high dose chemotherapy and/or irradiation for the purpose of
myeloablation, in preparation for stem cell or bone marrow transplantation.
Mucositis adversely impacts the quality of life of cancer patients in several
ways. For example, the mouth and throat sores of mucositis can cause
significant pain
and make it difficult to eat, drink, and even take oral medication. Mucositis
is also
accompanied by a severe risk of infection, as it can lead to a breach in the
otherwise
protective linings of the oral mucosa and gastrointestinal tract, which are
colonized by
a vast array of microorganisms. Further, efforts to counter the discomforts of
mucositis can lead to disruptions in cancer treatment, alterations in
treatment dosages,
or shifting to different modes of treatment. Severe mucositis can also lead to
the need
for parenteral nutrition or hospitalization. The development of effective
approaches to
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preventing and treating mucositis is therefore important for improving the
care of
cancer patients.
Summaryof the Invention
The invention provides methods of reducing the severity of oral or
gastrointestinal mucositis in patients. The methods include a step of
administering to
the patients a composition containing one or more compounds that block
activation of
toll-like receptor 4 (TLR4), such as a lipid A analog, which may be within the
formula:
RA O O O A'
A NH R CJNO I3 O ~R'
I 4 R2
where Rl is selected from the group consisting of:
0
0 OH
J K,
0
0 L)~ J
Q
0
0 Ll-k M-Q
J K
2
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0 01--, 4
J K
O 0 Q
\
J K
0
J G- K 5 and
0 0
J,--K K
where each J, K, and Q, independently, is straight or branched C1 to C15
alkyl; L is
0, NH, or CH2; M is 0 or NH; and G is NH, 0, S, SO, or SOZ;
R2 is straight or branched C5 to C15 alkyl;
R3 is selected from the group consisting of straight or branched C5 to C18
alkyl,
0
L'
A CH- CH-B
,
0
A CH=CD
I
B
A C ,C- B
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0
A E B CH= CH-D , and
0
A E B C C- D
where E is NH, 0, S, SO, or SO2; each A, B, and D, independently, is straight
or
branched C 1 to C 15 alkyl;
R4 is selected from the group consisting of straight or branched C4 to C20
alkyl, and
O1-11 W
U v
where each U and V, independently, is straight or branched C2 to C 15 alkyl
and W is
hydrogen or straight or branched Cl to C5 alkyl;
RA is R5 or R5-O-CH2-, R5 being selected from the group consisting of
hydrogen, J',
-J'-OH, -J'-O-K', -J'-O-K'-OH, and -J'-O-PO(OH)2, where each J' and K',
independently, is straight or branched C 1 to C5 alkyl;
R6 is selected from the group consisting of hydroxy, halogen, Cl to C5 alkoxy
and C1
to C5 acyloxy;
Al and A2, independently, are selected from the group consisting of
OH,
0
11
0- P- OH
I
OH
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0
11
0 Z O- P- OH
I
OH
0
I I
Z P- OH
I
oH and
o z C02H
where Z is straight or branched C l to C 10 alkyl; or a pharmaceutically
acceptable salt
or phosphate ester thereof. One aspect of the invention includes phosphate
esters of
the above-noted formula, wherein at least one of the hydroxyl groups of A' or
A2 can
be substituted to form a phosphate ester.
An example of a Lipid A analog that can be included in the compositions of
the invention is a compound having the following structure:
CH30 O O OPO(OH)2
O O
(CH2sCHs
(HO)20P0 NH HO H )~~
CHs(CH2)sY_'_~ O (CH2sCH3 OCH3
or a pharmaceutically acceptable salt or phosphate ester thereof.
In a more specific example, the compound is of the following structure:
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CH3O O O 0 oo11OPO(OHz)z
O O
\\'" . .,,",ir \'\'~.,. (CH~gCH3
(HO)ZOPO NH HO rH
CH3(CHz)s-~~~ O 0-~~ (CHasCHa
OCH3
or a pharmaceutically acceptable salt or phosphate ester thereof.
Patients that can be treated according to the invention include those who have
oral or gastrointestinal mucositis. In addition, patients who do not have, but
are at risk
of developing, oral or gastrointestinal mucositis can be treated according to
the
invention. In the latter group of patients, the treatment can inhibit or
prevent the
development of mucositis.
Examples of treatments that may cause or place a patient at risk of developing
oral or gastrointestinal mucositis are radiation therapy and chemotherapy, as
described
further elsewhere herein or in the background section. Patients that can be
treated
according to the invention thus include, for example, cancer patients, as well
as
patients that have recently been, will shortly be, or are currently subject to
treatment
with head or neck irradiation, or stem cell or bone marrow transplantation.
According to the methods of the invention, compositions used in the invention
can be administered to a patient prior to, concurrently with, or after a
treatment that
has induced or places the patient at risk of developing oral or
gastrointestinal
mucositis, or a combination of these approaches can be used. In an example,
the
composition is administered at the same time as, within 1-4 hours of, or on
the same
day as the treatment, and then for 1-3 (e.g., 1-2) days thereafter (e.g., 1-2
times per
day). Other examples of treatment regimens are provided below.
The compositions can be administered to patients by any acceptable manner
known in the art, including topically (e.g., by gel, rinse, lozenge, cream,
ointment, or
patch), by intravenous infusion, orally (e.g., by tablet, capsule, lozenge,
cream,
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ointment, or patch), rectally (e.g., by suppository, ointment, or enema), or
vaginally
(e.g., by cream, ointment, gel, or suppository). Also, treatment according to
the
invention can be carried out in combination with other approaches to treating
mucositis, including antimicrobial and palliative treatments, as is discussed
fi.u-ther
below.
In addition to the methods noted above, the invention also includes use of the
compositions and compounds described herein in the preparation of inedicaments
for
reducing the severity of oral or gastrointestinal mucositis. Such medicaments
can be
used to treat patients who already have mucositis, in an effort to reduce the
symptoms
of the condition (partially or fully), to prevent the condition from
worsening, and/or to
reduce the level of worsening of the condition. The medicaments can also be
used
with patients who do not yet have, but are at risk of developing, mucositis.
As is
discussed elsewhere herein, such patients include cancer patients who are
scheduled to
receive, are currently receiving, or have previously received cancer treatment
involving radiation and/or chemotherapy. In patients such as these,
administration of
the medicaments can be carried out to reduce the severity of mucositis, to
inhibit its
development, or to prevent mucositis from occurring. Further, the compounds
included in such medicaments can be any of those made reference to elsewhere
herein,
as well as compounds falling within the formulae provided elsewhere herein. A
specific example of such a compound is as follows:
CH3O O 0 0 OPO(OH)2
O O
(CHZ)yCH3
(HO)ZOPO NH HO H Nj-"-~ CH3(CH2)6
~~ O
O (CHz)sCHa
IY '~
OCH3
or a pharmaceutically acceptable salt or phosphate ester thereof.
Further, the invention includes compositions including the compounds
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described herein, formulated for administration for reducing the severity of
mucositis
as described herein. As is described in detail below, these compositions can
include
the compounds in formulations such as gels for topical administration, rinses,
tablets,
capsules, chewing gum, lozenges, creams, ointments, enemas, suppositories, or
patches.
The invention provides several advantages. For example, in providing
approaches to reducing the severity of mucositis, an uncomfortable side effect
of
treatments such as radiation and chemotherapy, the methods of the invention
can
contribute to the well being of patients as they face the challenges of such
treatments.
Further, the methods of the invention can decrease the incidence of infection,
which is
a common consequence of mucositis. In addition, in providing increased comfort
to
patients, the methods of the invention can lead to increased compliance of
patients
with their therapeutic regimens, and also can contribute to increasing the
speed of
their recovery.
Other features and advantages of the invention will be apparent from the
following detailed description, the drawings, and the claims.
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Brief Description of the Drawings
Figure 1 is a graph showing the percent weight change of C3H/HeOuJ and
C3H/HeJ mice after snout irradiation treatment. Animals were weighed daily,
the
percent weight change from day 0 was calculated, and group means and standard
errors of the mean (SEM) calculated for each day.
Figure 2 is a graph showing the area under the curve (AUC) calculated for the
percent weight change exhibited by snout irradiation-treated C3H/HeOuJ and
C3H/HeJ mice. This calculation was made using the trapezoidal rule
transformation.
Group means were calculated and are shown with error bars representing SEM for
each group. A one-way Anova test showed a statistically significant difference
between the groups (P=0.008).
Figure 3 is a graph showing the mean serum IL-6 concentration of snout
irradiation-treated C3H/HeOuJ and C3H/HeJ mice measured by ELISA analysis at
the
indicated time points.
Figure 4 is a graph showing the mean serum TNF-a concentration of snout
irradiation-treated C3H/HeOuJ and C3H/HeJ mice measured by ELISA analysis at
the
indicated time points.
Figure 5 is a graph of epithelial histology scores for snout irradiation-
treated
C3H/HeOuJ and C3H/HeJ mice. Each sample was scored on a scale of 0-3 for
epithelial cell layer damage.
Figure 6 is a graph of connective tissue histology scores for snout
irradiation-
treated C3H/HeOuJ and C3H/HeJ mice. Each sample was scored on a scale of 0-3
for
connective tissue damage.
Figure 7 is a graph showing the mean numbers of inflammatory cells of snout
irradiation-treated C3H/HeOuJ and C3H/HeJ mice measured at the indicated time
points.
Figure 8 is a graph showing the mean numbers of mitoses in the epithelial cell
layer of snout irradiation-treated C3H/HeOuJ and C3H/HeJ mice measured at the
indicated time points.
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Figure 9 is a graph showing the mean numbers of blood vessels per 10 high
power fields of snout irradiation-treated C3H/HeOuJ and C3H/HeJ mice measured
at
the indicated time points.
Figure 10 is a graph showing the mean number of large blood vessels as a
percentage of total blood vessels per 10 high power fields for C3H/HeOuJ and
C3H/HeJ mice measured at the indicated time points. The values are expressed
as a
percentage of the total numbers of blood vessels observed in those fields.
Figure 11 is a graph showing percent weight change of C3H/HeOuJ mice
treated with the indicated amounts of eritoran, after snout irradiation
treatment. The
animals were weighed daily, the percent weight change from day 0 was
calculated,
and group means and standard errors of the mean (SEM) calculated for each day.
Figure 12 is graph showing the area under the curve (AUC) calculated for the
percent weight change exhibited by snout irradiation-treated C3H/HeOuJ mice
shown
in Figure 11. This calculation was made using the trapezoidal rule
transformation.
Group means were calculated and are shown with error bars representing SEM for
each group. A one-way Anova test showed no statistically significant
differences
between groups (P=0.261).
Figure 13 is a graph showing the minimum number of epithelial cell layers on
the dorsal surface of the tongue for snout irradiation-treated C3H/HeOuJ mice
treated
with the indicated amounts of eritoran, at the indicated timepoints.
Figure 14 is a graph showing the maximum number of epithelial cell layers on
the dorsal surface of the tongue for snout irradiation-treated C3H/HeOuJ mice
treated
with the indicated amounts of eritoran, at the indicated timepoints.
Figure 15 is a graph showing the minimum number of epithelial cell layers on
the ventral surface of the tongue for snout irradiation-treated C3H/HeOuJ mice
treated
with the indicated amounts of eritoran, at the indicated timepoints.
Figure 16 is a graph showing the maximum number of epithelial cell layers on
the ventral surface of the tongue for snout irradiation-treated C3H/HeOuJ mice
treated
with the indicated amounts of eritoran, at the indicated timepoints.
Figure 17 is a graph showing the percent weight change of animals treated
with eritoran (E5564) under the indicated regimens, as well as un-irradiated
and
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placebo controls. Animals were weighed daily, the percent weight change from
day 0
was calculated, and group means and standard errors of the mean (SEM)
calculated
for each day.
Figure 18 is a graph showing the area under the curve (AUC) calculated for the
percent weight change exhibited by each animal in the study. This calculation
was
made using the trapezoidal rule transformation. Group means were calculated
and are
shown with error bars representing SEM for each group. A single asterisk
signifies a
statistically significant difference between a group receiving radiati'on and
the un-
irradiated controls, two asterisks indicate a statistically significant
difference between
the group treated with eritoran on days 0-3, and the placebo controls
(irradiated)
(P=0.030).
Figure 19 is a graph showing the percent weight change of animals treated
according to the regimens indicated in the figure. Data are shown for animals
surviving until the end of the study only. Animals were weighed daily, the
percent
weight change from day 0 was calculated, and group means and standard errors
of the
mean (SEM) calculated for each day.
Figure 20 is a graph showing the area under the curve (AUC) calculated for the
percent weight change exhibited by each animal treated, according to the
regimens
noted in the graph. This calculation was made using the trapezoidal rule
transformation. Group means were calculated and are shown with error bars
representing SEM for each group. A single asterisk signifies a statistically
significant
difference between a group receiving radiation and the un-irradiated controls,
two
asterisks indicate a statistically significant difference between the group
treated with
eritoran on days 0-3, and the placebo controls (irradiated) (P=0.041).
Figure 21 is a graph showing mean epithelial scores and standard errors of the
mean for each of the indicated groups.
Figure 22 is a graph showing mean connective tissue scores and standard
errors of the mean for each of the indicated groups.
Figure 23 is a graph showing mean inflammation scores and standard errors of
the mean for each of the indicated groups.
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Figure 24 is a graph showing mean number of mitoses per 10 hpf and standard
errors of the means for each of the indicated groups.
Figure 25 is a graph showing the mean percent ulceration and standard error of
the mean for each of the indicated groups.
Figure 26 is a graph showing the mean number of inflammatory cells per 10
hpf and standard errors of the means for each of the indicated groups.
Figure 27 is a graph showing the percentage of the infiltrating inflammatory
cells that were neutrophils for each sample and the mean and standard
deviation for
each of the indicated groups.
Figure 28 is a graph showing the percentage of the infiltrating inflammatory
cells that were lymphocytes for each sample and the mean and standard
deviation for
each of the indicated groups.
Figure 29 is a graph showing the percentage of the infiltrating inflammatory
cells that were monocytes or macrophage for each sample and the mean and
standard
deviation for each of'the indicated groups.
Figure 30 is a graph showing the number of small blood vessels per 10 hpf and
the mean and standard errors of the means for each of the indicated groups.
Figure 31 is a graph showing the number of medium blood vessels per 10 hpf
and the means and standard errors of the means for each of the indicated
groups.
Figure 32 is a graph showing the number of large blood vessels per 10 hpf and
the means and standard errors of the means for each of the indicated groups.
Figure 33 is a graph showing the number of mast cells per 10 hpf and the
means and standard errors of the means for each of the indicated groups.
Figure 34 is a graph showing serum TNF-a levels measured using an ELISA
assay and the mean and standard error of the mean for each of the indicated
groups.
Figure 35 is a graph showing serum IL-6 levels measured using an ELISA
assay and the mean and standard error of the mean for each of the indicated
groups.
Figure 36 is a graph showing serum SAA levels measured using an ELISA
assay and the mean and standard error of the mean for each of the indicated
groups.
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Detailed Description
The present invention provides methods of reducing the severity of oral or
gastrointestinal mucositis. The methods can be used to treat patients who
already
have mucositis. In addition, the methods can also be carried out with patients
who do
not have, but are at risk of developing mucositis (e.g., cancer or other
patients
scheduled to receive, currently receiving, or previously treated with
radiation and/or
chemotherapy). In the latter group of patients, which do not yet have
mucositis,
treatment according to the invention can reduce the severity of mucositis
resulting
from their cancer treatment, inhibit the development of mucositis, or prevent
mucositis.
The invention is based on the discovery that blocking activation of toll-like
receptor 4 (TLR4) provides beneficial therapeutic effects in the reduction of
severity
of mucositis, as described herein. TLR4 is a receptor for endotoxin, or
lipopolysaccharide (LPS), which is shed from the cell walls of growing and
dying
bacteria and has been associated with the induction of inflammatory responses.
According to the present invention, TLR4 receptor activation is blocked by
administration of a TLR4 antagonist, leading to beneficial effects in the
reduction of
severity of mucositis. In addition to blocking endotoxin, treatment according
to the
invention may block the effects of heat shock proteins (HSP's) in mucositis.
In
particular, such proteins, which are stress inducible proteins, may be induced
during
stress including radiation therapy and chemotherapy. HSP60, 70, or 90 may be
endogenous ligands of TLR4, and thus may play a role in the mucositis induced
by
radiation therapy.
TLR4 antagonists used in the methods of the invention can be, for example,
analogs of the lipid A region of LPS, such as lipid A analogs that are within
the
formula set forth above, in the Summary of the Invention. An example of a
Lipid A
analog that can be included in the compositions of the invention is a compound
having
the following structure:
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CH3 OPO(OH)Z
0 O
HO)20P NH H N (CH2)9CH3
H
CHs(CHz1s~ 0,,'.'~(CH2)6CH3
OCH3
or a pharmaceutically acceptable salt or phosphate ester thereof.
In a more specific example, the compound is of the following structure:
CHsO 0 0 ~~pOPO(OHz)z
O O
(HO)ZOPO~O ~~~~NH HO~~~~ (CH2)9CH3
H
CH3(CH2)6\,/ ,,/ O 0-1~1~ (CHz)sCHs
OCH3
or a pharmaceutically acceptable salt or phosphate ester thereof. This
compound, is
known as eritoran (also known as compound E5564, compound 1287, and SGEA) and
is described in U.S. Patent No. 5,935,938.
Other examples of compounds that can be used in the invention include the
following:
CH3OO O O ,OPO(OH)z
O OH
HO)zOPe ='"NH HO'l. =.,HA/ ~(CHz)joCHs
CHa(CHz)s O O ON,,,~ (CHz)sCHs
OCH3
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CH3O O O O 'OPOO(OH)2
,~
(HO)~OPO'~~. .,~NH HO'~~. .,,H)~, (CH2)12CH3
CH3(CH2)6 O O O,,.,~ (CH2)6CH3
IOH
CH3O O 0 O ,%OPO(OH)2
O O
(HO)20P0'%%, s,~NH HO'~~. H~(CH2),oCH3
N CH3(CH2)6 T\~/O O (CH2)6CH3
OH _
CH3O O O O % OPO(OH)2
O O
(HO)2OP0'~~. ,'NH HO'~~ H~(CH2)10CH3
CH3(CH2)6O~ (CH2)6CH3
OH (CH2)15CH3
CH3O O 0 O ,,OPO(OH)2
0 OH
(HO)2OP0'~~1.,~NH HO%~. .,~H(CHZ)loCFl3
CH3(CH2)6 O O (CH2)6CHa
OTH
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CH3O O 0 0 ,,OPO(OH)2
O OCH3
(HO)zOPOe JNH He H~(CH2)loCH3
CH3(CH2)6 O O O,,'~, (CH2)6CH3
OCH3
and
CH30 O O O %OPO(OH)2
O OCH3
(HO)20P0'~~. ,~~NH H~
.~H(CH2)loCH3
CHa(CH2)6 O O (CH2)sCH3
OH
and a pharmaceutically acceptable salt or phosphate ester thereof.
Additional TLR4 antagonists that can be used in the invention include, for
example, compound B531 (U.S. Patent No. 5,530,113), as well as other compounds
described in the following patents: U.S. Patent No. 5,935,938; U.S. Patent No.
5,612,476; U.S. Patent No. 5,756,718; U.S. Patent No. 5,843,918; U.S. Patent
No.
5,750,664; U.S. Patent No. 6,235,724; U.S. Patent No. 6,184,366; and U.S.
Patent No.
5,681,824. Methods for making these compounds are also described in these
documents. Additional methods for making such drugs are described, for
exainple, in
WO 02/94019.
According to the methods of the invention, a TLR4 antagonist is administered
to a patient before, during, and/or after treatment with a therapy that causes
oral or
gastrointestinal mucositis or puts the patient at risk of developing such
mucositis. As
is noted above, such treatments include radiation and chemotherapy, which act
by
blocking the growth of rapidly dividing cells, such as cancer cells and
epithelial cells
that line the surfaces of the gastrointestinal, respiratory, and genitourinary
tracts.
Specific examples of treatments that can lead to mucositis include radiation
treatment
(e.g., head and/or neck, whole body, targeted, and/or hyperfractionated
radiation), as
well as chemotherapeutic regimens used in the treatment of, or as adjuvant
treatments
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for, conditions such as breast cancer, colon cancer, gastric cancer,
genitourinary (e.g.,
bladder, prostate, or testicular) cancer, gynecologic (e.g., cervical,
endometrial,
ovarian, or uterine) cancer, head and neck/esophageal cancer, leukemia, lung
(small
cell or non small-cell) cancer, lymphoma (Hodgkin's or non-Hodgkin's),
melanoma,
multiple myeloma, pancreatic cancer, and sarcoma.
As is known in the art, cancers such as these can be treated using approaches
involving immunotherapy by use of agents such as, for example, rituximab,
cetuximab, or bevacizumab, alone or in combination with chemotlierapy or
radiation
therapy. In other examples, chemotherapeutic approaches that may induce
mucositis
include those utilizing (either as single agents or in combinations) platinum
derivatives such as carboplatin, cisplatin, and oxaplatin; mitosis inhibitors
such as
paclitaxel, docetaxel, vinorelbine, vincristine, and vinblastine;
topoisomerase
inhibitors such as etoposide, irinotecan, and topotecan; antimetabolites such
as
gemcitabine, capecitabine, fludarabine, methotrexate, 5-fluorouracil,
cladribine,
pentostatin, and cytarabine; DNA synthesis inhibitors such as doxorubicin,
epirubicin,
idarubicin, daunorubicin, bleoniycin, mechlorethamine, and mitoxantrone;
alkylating
agents such as cyclophosphamide, ifosfamide, and melphalan carmustine;
hormonal
oncologics such as estramustine; and agents having other or unknown mechanisms
such as dacarbazine. Use of these and other approaches to treating cancer is
well
known to those of skill in the art.
TLR4 antagonists such as those noted above can be administered using
standard methods including, for example, topical approaches and intravenous
infusion. The particular approach and dosage used for a particular patient
depends on
several factors including, for example, the type of cancer treatment, the
location(s) of
any discomfort, and the general health of patient. Based on factors such as
these, a
medical practitioner can select an appropriate approach.
Treatment according to the invention can begin prior to cancer treatment
(e.g.,
1-2 days or up to 1 week prior to cancer treatment), at or near the same time
as cancer
treatment (e.g., simultaneously with, within 1-4 hours of, or on the same day
as cancer
treatment), or shortly after the cessation of cancer treatment (e.g., within 1-
4 days of
cessation, and/or prior to or upon appearance of symptoms). Treatment can then
be
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maintained, for example, until any symptoms of mucositis have substantially
cleared
or the risk of developing such symptoms has passed. Thus, treatment started
before or
at or near the same time as cancer treatment can be maintained, e.g., for 1-3,
e.g., 1-2
days. In other examples, treatment is maintained for 1-4 or 2-3 weeks
following the
cessation of cancer treatment, as determined to be appropriate by one of skill
in the
art. In specific examples, the treatment according to the present invention is
carried
out prior to cancer treatment only; prior to and concurrently with cancer
treatment
only; prior to, concurrently with, and after cessation of cancer treatment;
concurrently
with cancer treatment only; concurrently with and after cessation of cancer
treatment
only; after cessation of cancer treatment only; or prior to and after
cessation of cancer
treatment only. Further, treatment according to the methods of the invention
can be
altered, stopped, or re-initiated in a patient, depending on the status of any
symptoms
of mucositis. Treatment can be carried out at intervals determined to be
appropriate
by those of skill in the art. For example, the administration can be carried
out 1, 2, 3,
or 4 times/day.
In the case of patients having or at risk of developing mucositis in the oral
cavity, a TLR4 antagonist, as described herein, can be administered to the
oral cavity
in the form of a gel, paste, spray, cream, ointment, or patch that is applied
to affected
or at risk areas. Such patients can also be treated by the use of an oral
rinse, chewing
gum, or lozenge including the drug. The drug can be administered to patients
affected
in rectal or vaginal areas by use of formulations in the form of gels, creams,
ointments, suspensions, or suppositories. Further, administration can be by
use of an
enema. In another example, in the case of patients affected in the nasal
cavity, the
drug can be administered by topical administration, as described herein, or by
inhalation of the drug (see, e.g., U.S. Patent No. 6,683,063). In other
approaches, the
drug can be administered by injection (e.g., local injection), or by infusion
(intravenous or intra-arterial), as discussed fi-ther below.
Formulation of drug compounds for use in the modes of administration noted
above (and others) are known in the art and are described, for example, in
Renzington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990,
Mack
Publishing Company, Easton, PA (also see, e.g., M. J. Rathbone, ed., Oral
Mucosal
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WO 2007/031879 PCT/IB2006/003538
Drug Delivery, Drugs and the Pharm.aceutical Sciences Series, Marcel Dekker,
Inc.,
N.Y., U.S.A., 1996; M. J. Rathbone et al., eds., Modified-Release Drug
Delivery
Technology, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc.,
N.Y., U.S.A., 2003; Ghosh et al., eds., Drug Delivery to the Oral Cavity,
Drugs and
the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 2005;
and
Mathiowitz et al., eds., Bioadhesive Drug Delivery Systems, Drugs and the
Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1999.
All patients, and in particular those affected (or at risk) in internal
regions that
are not readily accessible for topical administration, can be treated by a
systemic
approach, such as intravenous infusion. This approach to administration may be
particularly convenient in the case of patients who already have a catheter in
place for
the administration of chemotherapeutic or other drugs. Examples of such
approaches,
in which the drug administered is eritoran (see above) and the indicated
amounts of
the drug are based on an assumed average weight of a subject of 70 kg, are as
follows.
In a first example, the drug can be administered at a low dosage by continuous
intravenous infusion. As a specific example, the drug can be administered
continuously at a rate of 10-500 (e.g., 50-400 or 100-200) g/hour over the
course of
the treatment. In another example, in which a patient requires longer-term
care, the
drug can be administered intermittently (e.g., every 12-24 hours) at a dosage
of, for
example, 0.1-20 (e.g., 1-8, 2-7, 3-6, or 4-5) mg/hour for 2-6 (e.g.,
approximately 4)
hours. In a variation of this approach, the initial or loading dose is
followed by
maintenance doses that are less than (e.g., half) the loading dose or by
continuous
infusion as described above in the first example. The duration of such
treatment can
be determined by those of skill in the art, based on factors such as, for
example, the
severity of the condition and the observation of improvements. Additional
details
concerning the use of infusion to administer TLR4 antagonists, such as
eritoran, are
provided in US-2003-0105033-A1 (bolus or intermittent infusion) and WO
00/41703
(continuous infusion), the contents of each of which are incorporated herein
by
reference.
When administering the compound eritoran by intravenous infusion, it is
preferable to use devices and equipment (e.g., catheters, such as central or
peripheral
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WO 2007/031879 PCT/IB2006/003538
venous catheters, tubing, drip chambers, flashback bulbs, injection Y sites,
stopcocks,
and inf-usion bags) that are compatible with the drug. In particular,
catheters including
a chlorhexidine-based antimicrobial coating have been found to disrupt the
size of the
micelles of the drug that are formed during formulation, leading to inadequate
concentrations in blood. Thus, it is preferable to use devices and equipment
that have,
for example, a non-chlorhexidine-based antimicrobial coating, such as an
antimicrobial coating that includes one or more other antibiotics, such as
rifampin or
minicyclin.
The invention also includes kits that include one or more TLR4 antagonists
(e.g., a Lipid A analog as described above, e.g., the compound eritoran) and
instructions to use the drug in the methods described herein. The kits can
also
optionally include devices or equipment used in administration (e.g., a
catheter
lacking a chlorhexidine coating) and/or a solution for administering the drug,
such as
a 5% dextrose (e.g., glucose) solution.
The methods of the invention can be used alone or in conjunction with other
approaches to reducing the severity of mucositis. For exa.inple, the methods
of the
invention can be carried, out in combination with antimicrobial or antifungal
therapies,
e.g., therapies involving administration of antibiotics such as nystatin,
amphotericin,
acyclovir, valacyclovir, clotimazole, and fluconazole. As a specific example
of such
treatment, patients with head and neck cancer receiving radiotherapy have
colonization of the oropharyngeal region with gram-negative bacteria.
Selective
decontamination of the oral cavity with anti-microbial agents has the benefit
of
reducing oral mucositis associated with radiation therapy, but there may be
limitations
to the beneficial effects of such treatment. Anti-microbial therapy can kill
bacteria,
but cannot reduce endotoxin, and indeed may actually increase endotoxin. As
endotoxin is a potent mediator of inflammation, it may contribute to the
aggravation
of mucositis and, thus, co-treatment with an antiendotoxin compound (e.g., a
Lipid A
analog, such as eritoran) and antibiotics can be used as a more effective
approach to
treating oral mucositis in such patients, according to the invention.
The methods of the invention can also be used in conjunction with palliative
therapies including the use of topical rinses, gels, or ointments that include
lidocaine,
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WO 2007/031879 PCT/IB2006/003538
articaine, and/or morphine, as well as other analgesic or anti-inflammatory
agents.
Specific examples of other agents and approaches that can be used in
combination
with TLR4 antagonists, according to the methods of the invention, include the
following: palifermin (recombinant keratinocyte growth factor; rHuKGF;
KepivanceTM; Amgen) and AES-14 (uptake-enhanced L-glutamine
suspension)(Peterson, J. Support Oncol. 4(2 Suppl. 1)9-13, 2006); oral
cryotherapy,
low-level laser therapy, chlorhexidine, amifostine, hematologic growth
factors,
pentoxifylline, and glutamine (Saadeh, Pharmacotherapy 25(4):540-554, 2005);
amifostine, antibiotic paste or pastille, hydrolytic enzymes, ice chips,
benzydamine,
calcium phosphate, honey, oral care protocols, povidone, and zinc sulphate
(Worthington et al., Cochrane Database Syst. Rev. 2:CD000978, 2006);
flurbiprofen
(e.g., administered as a tooth patch; Stokman et al., Support Care Cancer
13(1):42-48,
2005); diphenhydramine, magnesium hydroxide/aluminum hydroxide, nystatin, and
corticosteroids (Chan et al., J. Oncol. Pharm. Pract. 11(4):139-143, 2005);
oral
transmucosal fentanyl citrate (e.g., administered in the form of a lozenge;
Shaiova et
al., Support Care Cancer 12(4):268-273, 2004); clonazepam (e.g., in the form
of a
tablet; Gremeau-Richard et al., Pain 108(102):51-57, 2004); capsaicin (e.g.,
in the
form of a lozenge; Okuno et al., J. Cancer Integr. Med. 2(3):179-183, 2004);
ketamine
(e.g., in the form of an oral rinse; Slatkin et al., Pain Med. 4(3):298-303,
2003); and
granulocyte-macrophage colony-stimulating factor (GM-CSF)/granulocyte colony-
stimulating factor (G-CSF), laser light therapy, and glutamine supplements
(Duncan et
al., Aliment. Pharmacol. Ther. 18(9):853-874, 2003).
The present invention is based, in part, on the following experimental
results.
EXAMPLE I
1. INTRODUCTION
1.1 Rationale
Two strains of C3H mice (C3H/HeJ and C3H/HeOuJ) differ from one another
by the presence or absence of the LPS receptor TLR4 (present in the C3H/HeOuJ
strain). C3H/HeJ mice are more sensitive to the lethal effects of total body
radiation,
but do not develop oral mucositis to the same extent as do the C3H/HeOuJ mice
after
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a localized acute radiation to the snout. The mechanistic basis for these
differences is
not understood.
1.2 Acute Snout Radiation Model
The acute mouse snout radiation model in mice has been used to determine the
radioprotective properties of experimental compounds. The course of oral
mucositis
in this model is well defined and results in peak mucositis 10-12 days
following
radiation. The acute model has little systemic toxicity, resulting in few
radiation-
induced animal deaths. In the present study, we used a dose of 30 Gy to induce
oral
mucositis.
2. STUDY OBJECTIVE AND SUMMARY
2.1 Study Objective
The objective of the study described below was to evaluate the effect of
localized acute radiation on the severity and duration of oral mucositis on
two strains
of mice. Wild type C3H/HeOuJ mice were compared to the endotoxin resistant
strain
C3H/HeJ. Mucositis was induced using an acute radiation dose of 30 Gy directed
to
the mouse snout. At several time points after radiation, groups of four mice
of each
strain were sacrificed. At the time of sacrifice, the tongues were removed and
dissected into three pieces. The anterior third of each tongue was fixed in
formalin for
subsequent histological analysis. The middle third of each tongue was
extracted to
provide mRNA for analysis of cytokine expression levels. The posterior portion
of
each tongue was flash frozen in liquid nitrogen for future analysis. At the
time of
sacrifice, blood was taken from each animal and serum was prepared for
subsequent
cytokine analysis. This study focused on the pro-inflammatory cytokines TNF-a
and
IL-6.
2.2 Study SummarX
A total of sixty-four (64) mice were used. Fifty-six (56) mice (28 each
C3H/HeOuJ and 28 C3H/HeJ) were given a single dose of 30 Gy radiation directed
to
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the snout on day 0. In addition, eight (8) mice (4 C3H/HeOuJ and 4 C3H/HeJ)
were
used as the no radiation control animals. Animals were sacrificed and blood
and
tissue taken according to the schedule described in Table 1.
Hour / Da
0 2H6H 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Randomize animals X
W e i h, R e c o r d survival X X X X X X X X X X X X X X X
Irradiate all animals 30 to snout X
Sacrifice 4 anirnalstgroup 1 and 2) X X X X X X X
dissect oral mucosa
Sacrifice 4 animaistgroup 3 and 4) X
dissect oral mucosa
Obtain serum from each anima
Table 1. Histological and cytokine comparison of the effects of ionizing
radiation of the
oral mucosa of C3H/HeJ and C3H/HeOuJ mice
3. STUDY DESIGN
Sixty-four (64) mice (32 C3H/HeOuJ and 32 C3H/HeJ) were used. The mice
were randomized into four (4) groups of either 28 animals (groups 1 and 2),
the
radiated groups, or 4 animals (groups 3 and 4), the un-irradiated control
groups, as
described in Table 2.
Mouse
Group Strain N Radiation
30 Gy
I C3H/HeOuJ 28 YES
WT
2 C3H/HeJ 28 YES
Mutant
3 C3H/HeOuJ 4 NO
WT
4 C3H/HeJ 4 NO
Mutant
Table 2. Animal allocation by experimental group.
Every day for the period of the study (day 0 to day 14), each animal was
weiglled. Animals in groups 1 and 2 received a single dose of 30 Gy radiation
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WO 2007/031879 PCT/IB2006/003538
focused on the snout on day 0. A lead shield protected the remainder of the
animal
body. At 2 hours, 6 hours, 24 hours (1 day), 3 days, 6 days, 10 days, and 14
days after
radiation, 4 animals from groups 1 and 2 were sacrificed and blood and tissue
were
collected as described below. Animals in groups 3 and 4 were sacrificed, the
tongues
dissected, and blood collected on day 1. The tongues from each animal were
dissected
into 3 pieces (anterior, middle, and posterior) and each tongue was fixed in
formalin.
Mucositis was assayed by histological analysis of hematoxylin and eosin (H&E)
stained sections of the formalin fixed tongues. Mucositis scoring was done in
a
blinded manner according to a validated scale. Serum samples were assayed for
the
cytokines TNF-a and IL-6 using a standard ELISA assay.
4. MATERIAL AND METHODS
4.1 Animals
C3H/HeOuJ and C3H/HeJ mice (Jackson Laboratories), aged 5 to 6 weeks
with body weights of 22.3 g, were used. Animals were individually numbered
using
an ear punch and housed in small groups of approxiunately 5 animals per cage.
Animals were acclimatized prior to study commencement. During this period of
at
least 2 days, the animals were observed daily in order to reject animals that
presented
in poor condition.
4.2 Housin~
The study was performed in animal rooms provided with filtered air at a
temperature of 70 F +/-5 F and 50% +/-20% relative humidity. Animal rooms were
set to maintain a minimum of 12 to 15 air changes per hour. The room was on an
automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no
twilight.
Bed-O-Cobs bedding was used, and was changed a minimum of once per week.
Cages, tops, bottles, etc. were washed with a commercial detergent and allowed
to air
dry. Prior to use, these items were wrapped and autoclaved. A commercial
disinfectant was used to disinfect surfaces and materials introduced into the
hood.
Floors were swept daily and mopped a minimum of twice weekly with a commercial
detergent. Walls and cage racks were sponged a minimum of once per month with
a
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dilute bleach solution. A cage card or label with the appropriate information
necessary to identify the study, dose, animal number, and treatment group was
placed
on all cages. The temperature and relative humidity was recorded during the
study,
and the records retained.
4.3 Diet
Animals were fed with a Labdiet 5001 chow and water was provided ad
libitum.
4.4 Animal Randomization and Allocations
Mice were randomly and prospectively divided into four (4) treatment groups
prior to irradiation. Each animal was identified by an ear punch corresponding
to an
individual number. A cage card was used to identify each cage or label marked
with
the study number, treatment group number, and animal numbers.
4.5 Radiation
Machine calibration was checked within two weeks of the onset of the study.
A single dose of radiation (30 Gy/dose) was administered to all animals in
groups 1
and 2 on day 0. Radiation was generated with a 160 kilovolt potential (15-ma)
source
at a focal distance of 50 cm, hardened with a 0.35 mm Cu filtration system.
Irradiation was done at a rate of 121.5 cGy/minute. Animals were anesthetized
prior
to radiation, and placed under lead shielding such that only the snout was
exposed
4.6 Tissue and Blood Collection and Analysis
4.6.1 Animal sacrifice and tissue collection
Animals in groups 3 and 4 were the un-irradiated control animals. The
measurements from these animals provided a baseline control for all the
irradiated
samples in this study. The 4 animals in each of groups 3 and 4 were sacrificed
on day
1.
Animals from groups 1 and 2 were sacrificed at several time points during the
course of the study. At each time point, 4 animals per group were sacrificed.
The
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time points were 2 hours, 6 hours, 24 hours, 3 days, 6 days, 10 days, and 14
days after
radiation.
At the time of sacrifice, the tongues were removed and dissected into three
pieces. The anterior third of each tongue was fixed in formalin for subsequent
histological analysis. The middle third of each tongue was extracted to
provide
mRNA for analysis of cytokine expression levels. The posterior section of each
tongue was flash frozen in liquid nitrogen and stored for future analysis.
At the time of sacrifice, approximately 1 mL of blood was taken from each
animal and serum was prepared for subsequent cytokine analysis. This study
focused
on the pro-inflammatory cytokines TNF-a and IL-6.
4.62 Cytokine ELISA
Enzyme linked immunosorbent assays (ELISAs) were performed for cytokines
TNF-a and IL-6 using kits purchased from R and D systems. These kits were used
in
accordance with the manufacturer's instructions. All determinations were made
in
duplicate on serum samples stored at -80 C. If insufficient serum had been
collected
to run both IL-6 and TNF-a, samples were diluted 1:2 or 1:4, and run in
duplicate in
both assays. All assays were performed using 50 gL of sample per well.
4.6.3 Histology
Histological samples were fixed in 10% formaldehyde in saline and process
for paraffm histology using standard techniques. Slides were stained with
hematoxylin and eosin (H&E) and reviewed by a board certified pathologist.
4.7 Assessment of Results
Statistical differences between treatment groups were determined using One
Way ANOVA. Body weights were evaluated for differences between the treatment
groups.
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5. RESULTS AND DISCUSSION
5.1 Survival
A total of 6 deaths occurred on day 10. These were equally distributed
between the C3H/HeOuJ and C3H/HeJ groups (3 deaths in each group) and, as a
result of this, only one animal in each group was sacrificed on day 14.
Additional
animals were subsequently irradiated in order to provide additional animals to
provide
data for the day 14 time-point.
5.2 Weights igures 1 and 2)
The mean percentage weight change for each group is shown in Figure 1. The
weight change data show that both groups of animals lost approximately 5% of
their
starting body weight by day post irradiation, then gained weight until day 6.
From day
6 until day 13, the C3H/HeJ mice maintained their weight between no gain and
5%
increase relative to their starting weight. The C3H/HeOuJ lost approximately
10% of
their body weight between days 7 and 9, and did not gain weight before day 13.
To
evaluate the differences between the two groups, the area under the curve
(AUC) for
each individual animal was calculated and the differences were evaluated using
a One-
Way ANOVA analysis. The mean AUC data is shown in Figure 2. The One-Way
ANOVA analysis showed that there was a statistically significant difference
between
the groups (P=0.008).
5.3 Serum Cytokine Levels
Serum levels of cytokines IL-6 and TNF-a were evaluated by ELISA.
5.3.1 Serum IL-6 Concentrations
In un-irradiated C3H/HeOuJ mice, the mean serum concentration of IL-6 was
1.0 pg/mL. This level increased to 88.8 pg/mL at 6 hours post radiation,
before falling
to 12.0 pg/mL on day 3 following radiation and increasing to a peak level of
122.7
pg/mL on day 6. Days 10 and 14 showed a gradual decline from the peak levels
seen
on day 6. In un-irradiated C3H/HeJ mice, the mean serum concentration of IL-6
was
13.8 pg/mL. All other readings were between 25 and 42 pg/mL with the exception
of
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the day 10 time-point, when serum IL-6 concentrations increased to 69.4 pg/mL.
These data are shown in Figure 3.
5.3.2 Serum TNF-a Concentrations
The mean serum TNF-a concentration in un-irradiated C3H/HeOuJ mice was
48.0 pg/mL. There were 2 peaks in serum TNF-a levels in these mice, one after
2
hours (168.7 pg/mL) and one at day 10 (410.2 pg/mL). At time-points between
these
2 peaks, serum TNF-a concentrations were close to the levels seen in un-
irradiated
C3H/HeOuJ mice (31.6 pg/mL to 87.2 pg/mL). On days 10 and 14, the levels were
lower than in the un-irradiated controls (5.7 pg/mL and 6.6 pg/mL
respectively). In
C3H/HeJ mice, un-irradiated control mice had mean serum TNF-a concentrations
of
109.3 pg/mL. Subsequent post-irradiation readings were generally lower than
this,
ranging from 14.2 pg/mL at 6 hours post radiation to 133.8 pg/mL on day 10.
These
data are shown in Figure 4.
5.4 Tongue Histolow
Parts of each tongue were processed for routine hematoxylin and eosin (H&E)
histology. These slides were then reviewed by a board certified pathologist
and
scored for epithelial and connective tissue pathology on a scale of 0-3,
epithelial
mitoses, percent ulceration, skeletal muscle damage, number of inflammatory
cells per
10 high powered fields (including differential cell type analysis), and the
number of
small, medium, and large blood vessels.
5.4.1 Histological Score
The epithelium and connective tissue regions of each sample were each given
separate scores. The scores for the epitlielium are shown in Figure 5. The
mean
epithelial histological score for C3H/HeOuJ mice that had not been irradiated
was 0,
and this was also the case for all post-radiation time points except day 1,
when the
mean score was 0.25, and days 6 and 10, when the mean score was 2. In C3H/HeJ
mice, the mean epithelial histological score was 0 at all time points except
day 6,
when the score was 0.75. The data for the mean connective tissue histological
scores
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is shown in Figure 6. The mean connective tissue histological score for
C3H/HeOuJ
mice that had not been irradiated was 2. This score dropped to 0 at 2 hours
post-
radiation, increased to a score of 1 on day 1 post radiation,-before dropping
to 0 on
day 3 and increasing to 1.5 on days 10 and 14. In C3H/HeJ mice, the mean
epithelial
histological score was 1.25 in mice that had not been irradiated, dropping to
0.25 at 2
hours post radiation before increasing gradually to 1.25 on day 10. In the
connective
tissue, the histological score was as high or higher in the control un-
irradiated mice
than at any time following radiation, in both C3H/HeOuJ and C3H/HeJ mice. The
reasons for this are currently unknown.
5.4.2 Inflamination
The mean number of inflammatory cells per ten high powered fields for each
strain of mouse at each time point was calculated and the results are shown in
Figure
7. The numbers of cells seen in the connective tissue of un-irradiated animals
was
higher than expected in both strains of mice, and was lower at all post
irradiation
time-points in the C3H/HeJ mice. In the C3H/HeOuJ mice, the numbers of
inflammatory cells seen at most time points was also lower than those observed
in un-
irradiated controls, except for day 10, when the number of cells was
approximately 2
times higher than the un-irradiated controls (and about 10 times higher than
the 2 hour
and 6 hour time-points) and on day 14, when the numbers observed were about
50%
higher than the un-irradiated controls. As for the connective tissue histology
scores,
the reasons for the unexpectedly high numbers seen in the un-irradiated
animals are
unknown. In all cases, the bulk of the infiltrate was composed of lymphocytes,
with
monocytes and macrophage accounting for ahnost all of the non-lymphocytic
cells
most animals. Significant numbers of polymorphonucleocytes (PMNs or
neutrophils)
were only seen in three animals from the day 10 time-point (1 OuJ and 2 HeJ).
5.4.3 Epithelial Cell Mitoses
The number of mitotic figures seen in the epithelial cell layer was counted
and
the mean number of mitoses per ten high power fields for each strain at each
time
point is shown in Figure 8. The number of mitotic figures counted in the
epithelial
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cell layer of C3H/HeOuJ mouse tongues was generally low, with a mean of 0.4 in
un-
irradiated mice and numbers lower than this at all time points except day 6,
when a
mean of 2.75 was observed. In C3H/HeJ mice the numbers of mitoses seen in un-
irradiated mice was lower than in C3H/HeOuJ mice with a mean of 0.1. However,
this increased to 0.4 by day 10 post radiation.
5.4.4 Blood Vessels
The number of blood vessels per ten high power fields was counted for each
sample and the mean number for each strain of mouse at each time point
calculated.
These data are shown in Figure 9. The number of blood vessels per 10 high
power
fields was 26.6 in un-irradiated C3H/HeOuJ mice and 27.2 in un-irradiated
C3H/HeJ
mice. In C3HIHeOuJ mice, the number of blood vessels had apparently dropped to
4.5 by 2 hours post radiation, rising to 20.6 on day 1, before falling to 8.5
on day 3,
and increasing on days 6 and 10, before reaching a peak of 33.7 on day 14.
This
represents an increase of 27% relative to the un-irradiated controls and 648%
relative
to the 2 hour time point. It is interesting to note that the un-irradiated
control animals,
sacrificed on day 1, have similar levels to the day 1 time-point. In the
C3H/HeJ mice,
the number of blood vessels was generally close to the un-irradiated controls,
reaching
a minimum of 18.3 at 2 hours post radiation, and a maximum of 33.1 on day 6
post
radiation. To evaluate the qualitative changes in the blood vessels, the
numbers of
large blood vessels per 10 high power fields were evaluated and the resulting
numbers
expressed as a percentage of the total number of vessels seen in the same 10
high
power fields. The results of this analysis are shown in Figure 10 and indicate
that the
number of large blood vessels seen in the C3H/HeOuJ mice increased from a mean
of
9.2% in the un-irradiated control mice, to a peak of 26.5% on day 1 (24 hours
post
radiation), and declined during the remainder of the study. The C3H/HeJ mice
had a
slightly higher control level of 13.2% in the un-irradiated mice, which
increased to a
peak of 18.7% on day 6 post radiation, and fell to levels below controls on
days 10
and 14 post radiation.
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6. CONCLUSIONS
1. C3H/HeOuJ mice showed greater weight loss than the C3H/HeJ mice
during this study, and the differences observed were statistically significant
when
evaluated with a one-way ANOVA test (P=0.008).
2. Analysis of serum cytokine levels showed that the un-irradiated C3H/HeJ
control mice had higher levels than their C3H/HeOuJ counterparts, but that the
C3H/HeOuJ mice showed greater increases in serum cytokines following radiation
than C3H/HeJ, with peak levels of both IL-6 and T'NF-a being seen on day 6
post
irradiation.
3. Histologically, very little change was seen in C3H/HeJ mice. C3H/HeOuJ
mice showed a significant disturbance of the epithelium on days 6 and 10
following
radiation. Histological scores for the connective tissue were high in the un-
irradiated
control C3H/HeOuJ mice and declined at 2 hours to 6 days post radiation,
returning to
near control levels at days 10 and 14 post radiation.
4. The numbers of inflammatory cells present showed little change in the
C3H/HeJ mice but increased to a peak on day 10 post radiation in C3H/HeOuJ
mice,
coinciding with the tissue peak tissue cytokine levels in these animals.
Infiltrates were
predominantly lymphocytic in nature.
5. The number of mitoses observed in the epithelial cell layer showed a slight
increase in C3H/HeJ mice, peaking on day 10, while a significant spike in
mitotic
activity was noted on day 6 in the C3H/HeOuJ mice.
6. In the analysis of the number and size of blood vessels observed, few
changes were noted in the C3H/HeJ mice, while the C3H/HeOuJ mice showed a
decrease in the number of blood vessels immediately following radiation (2 and
6
hours post radiation), combined with an overall increase at the later time
points (days
10 and 14 post radiation). An increase in the percentage of large blood
vessels was
noted 24 hours post radiation in C3H/HeOuJ mice.
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EXAMPLE II
1. INTRODUCTION
1.1 Rationale
As noted above in Example I, two strains of C3H mice (C3H/HeJ and
C3H/HeOuJ) differ from one another by the presence or absence of the LPS
receptor
TLR4 (present in the C3H/HeOuJ strain). In the experiments described above, it
is
established that the C3H/HeOuJ strain is susceptible to oral mucositis induced
by
focal radiation to the snout, while the C3H/HeJ strain is relatively resistant
to
radiation induced mucositis. Evaluation of the pro-inflammatory cytokines in
these
animals showed that the induction of these cytokines via the LPS receptor TLR4
in the
C3H/HeOuJ mice may play a role in the development of oral mucositis. The
purpose
of the study described below was to evaluate a compound that blocks
stimulation of
TLR4 (eritoran) in the murine model of oral mucositis.
1.2 Acute Snout Radiation Model
The acute mouse snout radiation model in mice has been used to determine the
radio-protective properties of experimental compounds. The course of oral
mucositis
in this model is well defmed and results in peak mucositis 10-12 days
following
radiation. The acute model has little systemic toxicity, resulting in
relatively few
radiation induced animal deaths. In this study, a dose of 30 Gy was used to
induce
oral mucositis.
2. STUDY OBJECTIVE AND SUMMARY
2.1 Study Objective
The objective of the study described below was to examine the effects of
eritoran administered subcutaneously on the severity and duration of oral
mucositis
induced by radiation. Mucositis is induced using an acute radiation dose of 30
Gy
directed to the mouse snout. At several time points after radiation, groups of
four
mice from each treatment group were sacrificed. At the time of sacrifice, the
tongues
were removed and dissected into three pieces. The anterior third of each
tongue was
fixed in formalin for subsequent histological analysis. The middle third of
each
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tongue was extracted to provide mRNA for analysis of cytokine expression, and
the
posterior portion of each tongue was flash frozen in liquid nitrogen and
stored for
future analysis. At the time of sacrifice, blood was taken from each animal
and serum
was prepared for subsequent cytokine analysis.
2.2 Study SuinmarX
A total of fifty-four (54) animals were used in this study. Forty-eight (48)
C3H/HeOuJ mice were divided into 3 groups of 16 animals per group (groups 1-
3).
An additional 6 animals were put into a separate control group (group 4) as
described
in Table 2.
3. STUDY DESIGN
Fifty-four (54) male C3H/HeOuJ mice aged 6-7 weeks and weighing
approximately 22 g were used. There were three (3) treatment groups of sixteen
(16)
animals each, and a control group of six (6) animals that received no
radiation. All
aninzals had a jugular cannula inserted into the left jugular vein on day -3.
Beginning
on day 0, animals in groups 1 and 4 were dosed twice a day by injection via
cannula
with placebo. Animals in group 2 were dosed with 2 IV injections of eritoran
at 1
mg/kg daily, starting 2 hours or less before radiation on day 0 and continuing
until day
10. Animals in group 3 were dosed with 2 IV injections of eritoran at 10 mg/kg
daily,
starting 2 hours or less before radiation on day 0 and continuing until day
10.
Animals in groups 1, 2, and 3 were given a single dose of 30 Gy radiation
directed to
the snout on day 0. The 6 animals in group 4 were used as the no radiation
control
animals (see Table 2). Eight animals in each of groups 1, 2, and 3 were
sacrificed and
blood and tissue taken according to the schedule described in Table 2.
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Table 2. Allocation by experimental group.
Group Number of Strain Treatment Radiation Sac points
animals
1 16 male C3H/HeOuJ placebo IV bid 30 Gy to 8 on day 6,
snout 8 on day 10
2 16 male C3H/HeOuJ eritoran 1mg/kg IV 30 Gy to 8 on day 6,
bid snout 8 on day 10
3 16 male C3H/HeOuJ eritoran 10 mg/kg 30 Gy to 8 on day 6,
IV bid snout 8 on day 10
4 6 male C3H/HeOuJ placebo IV bid none 6 on day 10
4. MATERIAL AND METHODS
4.1 Animals
C3H/HeOuJ mice (Jackson Laboratories), aged 5 to 6 weeks with body weight
of 21.3 g, were used. Animals were individually numbered using an ear punch
and
individually housed. Animals were acclimatized prior to study commencement.
During this period of at least 2 days, the animals were observed daily in
order to reject
animals that presented in poor condition.
4.2 Housin2
The study was performed in animal rooms as described above in section 4.2 of
Example I.
4.3 Diet
Animals were fed with Labdiet 5061 sterile irradiated rodent chow and water
was provided ad libitum.
4.4 Animal Randomization and Allocations
Mice were randomly and prospectively divided into three (3) treatment groups
prior to irradiation. Each animal was identified by an ear punch corresponding
to an
individual number. A cage card was used to identify each cage or label marked
with
the study number, treatment group number, and animal numbers.
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4.5 Radiation
Machine calibration was checked within two weeks of the onset of this study.
A single dose of radiation (30 Gy/dose) was administered to all animals in
groups 1
and 2 on day 0. Radiation was generated with a 160 kilovolt potential (15-ma)
source
at a focal distance of 50 cm, hardened with a 0.35 mm Cu filtration system.
Irradiation was done at a rate of 121.5 cGy/minute. Animals were anesthetized
prior
to radiation, and placed under lead shielding such that only the snout is
exposed
4.6 Tissue Collection and Analysis
4.6.1 Histology
Histological samples were fixed in 10% formaldehyde in saline and process
for paraffin histology using standard techniques. Slides were stained with
hematoxylin and eosin (H&E).
4.7 Assessment of Results
Statistical differences between treatment groups were determined using One
Way ANOVA. Body weights were evaluated for differences between the treatment
groups.
5. RESULTS AND DISCUSSION
5.1 Weights (Figures 11 and 12)
The mean percentage weight gain for each group for each day of the study is
shown in Figure 11. The un-irradiated control group gained an average of 8.1 %
during the study, as compared with a mean loss of 0.8% in the placebo group.
In the
groups receiving eritoran, a mean weight gain of 4.0% was seen in the group
receiving
1 mg/kg as compared with a net loss of 0.2% in the group receiving 10 mg/kg.
The
results of this analysis for the three groups receiving radiation are shown in
Figure 12.
There were no significant differences between these three groups (P=0.261).
When
compared against the un-irradiated controls, there were significant
differences
between the un-irradiated group and the radiated groups receiving placebo
(P<0.001)
and eritoran 10 mg/kg (P<0.001).
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5.3 Tongue Histology_
Each tongue was processed for routine hematoxylin and eosin histology.
Because of several technical reasons, a total of 44 samples were evaluated. Of
these
44 samples, 6 were in the un-irradiated control group, 12 were in the placebo
group (6
each on days 6 and 10), 14 were in the eritoran 1 mg/kg treated group (7 each
on days
6 and 10), and 12 were in the eritoran 10 mg/kg treated group (7 on day 6, and
5 on
day 10).
The most common overall observation over the entire data set was normal or
essentially normal. This was used in the description of 14 samples, 4 of which
were
in the un-irradiated control group. Normal was also used to describe 6 of the
14
samples in the 1 mg/mg eritoran treated group (2/7 samples from day 6 and 4/7
samples from day 10), and 3 of the 12 samples in the eritoran 10 mg/kg treated
group
(1/7 samples from day 6 and 2/5 samples from day 10). Only one of the 12
samples
from the placebo treated group was described as normal (a day 6 sample).
Hyperkeratosis was also seen in 14 of the samples, none of which were in the
un-
irradiated control group. Hyperkeratosis was most commonly seen in samples
from
the eritoran 10 mg/kg treated group, where it was applied to 7 of the 12
samples (3/7
at day 6 and 4/5 at day 10). Hyperkeratosis was seen in 5 of the 14 samples in
the
eritoran 1 mg/kg treated group (1/7 at day 6 and 4/7 on day 10). Only 2
sainples in the
placebo group were seen with hyperkeratosis, one at each time-point.
Epithelial
hyperplasia was seen in only 5 samples, however 4 of these samples were in the
eritoran 10 mg/kg treatment group (2 at each time-point) and the fifth was in
the
placebo group (day 6). These observations seem to indicate a substantial
improvement in both eritoran treatment groups relative to the placebo
controls, with
the high dose treatment group (10 mg/kg) showing a tendency to hyperplasia and
hyperkeratosis.
Connective tissue damage or disruption was seen in a total of 11 samples, 8 of
which were in the placebo treated group (3/6 on day 6 and 5/6 on day 10), 2
were in
the eritoran 1 mg/kg treated group (both day 6), 1 was in the eritoran 10
mg/kg treated
group (day 6). Loss or break in the epithelium was noted in 7 samples, and
epithelial
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atrophy was noted in an additional 5 samples. Of these 12 samples with
epithelial
damage, 5 were in the placebo treated group (2 on day 6 and 3 on day 10), 5
were in
the eritoran 1 mg/kg treated group (all on day 6), and 2 were in the eritoran
10 mg/kg
group (both on day 6). Increased cellularity was seen in 10 samples, 2 in the
placebo
group (one each on day 6 and day 10), 5 in the eritoran 1 mg/kg treated group
(one on
day 6 and 4 on day 10), and 3 in the eritoran 10 mg/kg treated group (one on
day 6 and
2 on day 10). Two types of infiltrate were observed, round cell or lymphocytic
infiltrates were noted in 8 samples, were evenly distributed among the groups
and
time-points, and were seen in one of the 6 un-irradiated controls. Mast cell
infiltrates
were observed in 9 samples, 7 of which were in the placebo treated group (5 on
day 6,
and 2 on day 10), and the other 2 samples were in the eritoran 10 mg/kg
treated group,
day 6 time-point. The other observations regarding vasodilation and increased
vascularity were evenly distributed or too rarely seen to show any meaningful
differences between the treatment groups. These observations indicate that
eritoran
treatment results in improved tongue histology, as shown by decreased
radiation-
induced connective tissue damage and mast cell infiltration.
5.3.1 Thickness of the Epithelial Surface on the Dorsal and Ventral Surfaces
of the
Tongue
Each sample was evaluated for the minimum and maximum number of
epithelial cell layers on the dorsal and ventral surfaces of the tongue. From
these
numbers, mean minimum and maximum thickness was calculated for each treatment
group at each time-point. For the dorsal surface of the tongue, the mean
minimum
number of cell layers in the un-irradiated controls was 6 cell layers. In the
placebo
control treatment group, the mean number of cell layers was 2.7 on day 6 and
2.0 on
day 10. In the eritoran treated groups, the mean minimum number of cell layers
was
2.8 (day 6) and 3.8 (day 10) in the 1 mg/kg group and 3.8 (day 6) and 3.3 (day
10) in
the 10 mg/kg group. These data are shown in Figure 13. The mean maximum
number of epithelial cell layers on the dorsal surface in the un-irradiated
controls was
8. In the placebo control treatment group, the mean number of cell layers was
4.8 on
day 6 and 3.5 on day 10. In the eritoran treated groups, the mean minimum
number of
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cell layers was 5.3 (day 6) and 6.4 (day 10) in the 1 mg/kg group, and 6.2
(day 6) and
6.1(day 10) in the 10 mg/kg group. These data are shown in Figure 14. On the
ventral
surface, the mean minimum number of cell layers in the un-irradiated controls
was 4
cell layers. In the placebo control treatment group, the mean number of cell
layers
was 1.7 on day 6 and 0.9 on day 10. In the eritoran treated groups, the mean
mininlum number of cell layers was 2.0 (day 6) and 2.8 (day 10) in the 1 mg/kg
group,
and 3.0 (day 6 and day 10) in the 10 mg/kg group. These data are shown in
Figure 15.
The mean maximum number of epithelial cell layers on the ventral surface in
the un-
irradiated controls was 6. In the placebo control treatment group, the mean
number of
cell layers was 4.2 on day 6 and 2.4 on day 10. In the eritoran treated
groups, the
mean minimum number of cell layers was 3.7 (day 6) and 4.6 (day 10) in the 1
mg/kg
group and 5.8 (day 6) and 5.9 (day 10) in the 10 mg/kg group. These data are
sliown
in Figure 16. These observations indicate that eritoran seems to protect the
epithelial
cell layer, with the 10 mg/kg group showing slightly greater protection that
the 1
mg/kg group, particularly on the ventral surface.
6. CONCLUSIONS
1. Significant mortality was seen during this study, but this excess mortality
was not associated with any one treatment group.
2. No statistically significant differences in weight gain were seen between
the
three irradiated treatment groups.
3. Both groups treated with eritoran showed improvement in tongue histology
relative to the placebo treated-control group, as determined by the number of
samples
described as normal, increases in epithelial hyperplasia and hyperkeratosis,
and
decreases in connective tissue damage and mast cell infiltrates.
4. Although both groups treated with eritoran showed improvements in tongue
histology, there were distinct differences in the descriptive histology
between the 1
mg/kg and 10 mg/kg groups, although it is not clear which dose showed the
greater
improvement.
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EXAMPLE III
1. INTRODUCTION
1.1 Rationale
As discussed above, two strains of C3H mice (C3H/HeJ and C3H/HeOuJ)
differ from one another by the presence or absence of the LPS receptor TLR4
(present
in the C3H/HeOuJ strain), and the C3H/HeOuJ strain are susceptible to oral
mucositis
induced by focal radiation to the snout, while the C3H/HeJ strain are
relatively
resistant to radiation induced mucositis. Further as described above,
evaluation of the
pro-inflammatory cytokines in these animals shows that the induction of these
cytokines via the LPS receptor (TLR4) in the C3H/HeOuJ mice may play a role in
the
development of oral mucositis. The experiments described in Example II
demonstrated the efficacy of eritoran in a model of oral mucositis. The study
described below identifies optimal dosing schedules for eritoran.
1.2 Acute Snout Radiation Model
The acute mouse snout radiation model has been used to determine the radio-
protective properties of experimental compounds. The course of oral mucositis
in this
model is well defined and results in peak mucositis 10-12 days following
radiation.
The acute model has little systemic toxicity, resulting in relatively few
radiation
induced animal deaths. In this study, a dose of 30 Gy was used to induce oral
mucositis.
2. STUDY OBJECTIVE AND SUMMARY
2.1 Study Objective
The objective of this study was to examine the effect of scheduling of
eritoran,
administered intraveneously, on the severity and duration of oral mucositis
induced by
radiation. Mucositis was induced using an acute radiation dose of 30 Gy
directed to
the mouse snout. At 10 days after radiation, groups of four mice from each
treatment
group were sacrificed. At the time of sacrifice, the tongues were removed and
fixed in
formalin for subsequent histological analysis. At the time of sacrifice, blood
was
taken from each animal and serum was prepared for subsequent cytokine
analysis.
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These samples were used for the measurement of serum Tumor Necrosis Factor
(TNF-
a), Interleukin-6 (IL-6), and Serum Amyloid A (SAA) levels.
2.2 Study Summar_y
Sixty (60) C3H/HeOuJ mice were obtained from Jackson Laboratories. These
animals were shipped with jugular cannulae already implanted. The animals were
randomly divided into 6 groups of 10 animals per group as described in Table
4.
3. STUDY DESIGN
Sixty (60) male C3H/HeOuJ mice aged 6-7 weeks and weighing
approximately 22 g were used. There were five (5) treatment groups of ten (10)
animals each, and a control group of ten (10) animals, which received no
radiation.
Beginning on day 0, 2 hours or less before radiation, animals in groups 1-6
were
dosed with either placebo or eritoran 10 mg/kg as detailed in Table 4. Dosing
continued twice daily from the day of radiation (day 0) until day 9. Animals
in groups
1 and 2 received placebo throughout the dosing period. Animals in group 3
received
eritoran at 10 mg/kg for the entire dosing period. Animals in group 4 received
eritoran at 10 mg/kg twice daily from day 0 until day 3, and then placebo
twice daily
until the end of the dosing period. Animals in group 5 received placebo twice
daily
from day 0 until day 2, then eritoran 10 mg/kg twice daily from day 3 until
day 6, and
then placebo twice daily until the end of the dosing period. Animals in group
6
received placebo twice daily from day 0 until day 5, then eritoran 10 mg/kg
twice
daily until the end of the dosing period. All drug and placebo administration
was via
intravenous via jugular cannula.
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Table 4. Allocation by experimental group
Group Number Treatment Eritoran Placebo Dose
of Volume
Animals
1 10 Male No Radiation Days 0-9 0.1 mL
Placebo
2 10 Male Placebo Days 0-9 0.1 mL
3 10 Male eritoran days 0-9 Days 0-9 0.1 mL
m bid
4 10 Male eritoran days 0-3 Days 0-3 Days 4-9 0.1 mL
10 m /k bid
5 10 Male eritoran days 3-6 Days 3-6 Days 0-2 & 0.1 mL
10m /k bid Days 7-9
6 10 Male eritoran days 6-9 Days 6-9 Days 0-5 0.1 mL
10 mg/kg bid
Every day for the period of the study (day 0 to day 10), each animal was
weighed to an
accuracy of 0.1 g. At 10 days after radiation, all animals were sacrificed and
the
tongues taken for histological analysis. Blood was taken at the time of
sacrifice and
5 serum was stored at -80 C.
4. MATERIAL AND METHODS
4.1 Animals
10 C3H/HeOuJ mice (Jackson Laboratories), aged 5 to 6 weeks with body
weights of 23.2 g, were used. Animals had jugular cannulas installed by
Jackson
Laboratories prior to shipment, and were individually numbered using an ear
punch
and individually housed. Animals were acclimatized prior to study
commencement.
During this period of at least 2 days, the animals were observed daily in
order to reject
animals that presented in poor condition.
4.2 Housin~
The study was performed in animal rooms as described above in section 4.2 of
Example I.
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4.3 Diet
Animals were fed with Labdiet 5061 sterile irradiated rodent chow and water
was provided ad libitunz.
4.4 Animal Randomization and Allocations
Mice were randomly and prospectively divided into three (3) treatment groups
prior to irradiation. Each animal was identified by an ear punch corresponding
to an
individual number. A cage card was used to identify each cage or label marked
with
the study number, treatment group number, and animal numbers.
4.5 Radiation
Machine calibration was checked within two weeks of the onset of this study.
A single dose of radiation (30 Gy/dose) was administered to all animals in
groups 1
and 2 on day 0. Radiation was generated with a 160 kilovolt potential (15-ma)
source
at a focal distance of 50 cm, hardened with a 0.35 mm Cu filtration system.
Irradiation was done at a rate of 121.5 cGy/minute. Animals were anesthetized
prior
to radiation, and placed under lead shielding such that only the snout is
exposed
4.6 Tissue Collection and Analysis
4.6.1 Histology
Histological sanlples were fixed in 10% formaldehyde in saline and processed
for paraffin histology using standard techniques. Slides were stained with
hematoxylin and eosin (H&E).
4.6.2 Cytokine ELISA
Enzyme linked immunosorbent assays (ELISAs) were performed for cytokines
TNF-a and IL-6 using kits purchased from R and D systems. Determination of
serum
amyloid A was performed using an ELISA kit from Biosource International. These
kits were used in accordance with the nlanufacturer's instructions. All
determinations
were made in duplicate on serum samples stored at -80 C. Samples were run in
duplicate in all three assays, and if insufficient serum had been collected to
run IL-6,
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SAA, and TNF-a assays, samples were diluted 1:4. All assays were performed
using
50 L of sample per well.
4.7 Assessment of Results
Statistical differences between treatment groups were determined using One
Way ANOVA. Body weights are evaluated for differences between the treatment
groups.
5. RESULTS AND DISCUSSION
5.1 Survival
A total of 108 cannulated animals were used in this study. Due to the limited
availability of the C3H/HeOuJ mice, these animals were processed in 3 groups
over a
period of 6 weeks. 57 of these mice survived until day 10. Of the 51 mice that
did
not survive until day 10, 21 , died or were euthanized on day 0, 11 due to
anesthesia
and radiation related issues, and 10 due to problems with the cannula (died
after initial
injection due to presumed clot, cannula not patent, or cannula pulled out). Of
the
remaining 30 animals that died or were enthanized during the study, 2 died on
day 1, 5
on day 2, 4 on day 3, 7 on day 4, 3 each on days 5 and 6, 1 each on days 7 and
8, and 2
each on days 9 and 10. The distribution of deaths by group was relatively
equal. Nine
(9) deaths were observed in each of the un-irradiated control group and the
vehicle
control group., Seven (7) deaths were observed in each of the groups treated
with
eritoran 10 mg/kg from days 0-10 or days 0-3. Nine deaths were observed in the
group treated with eritoran 10 mg/kg, from day 3 until day 6, and 10 deaths
were
observed in the group treated with eritoran 10 mg/kg, from day 6 until day 9.
5.2 Wei ts (Figures 17, 18, 19, and 20)
The mean percentage weight gain for each group for each day of the study is
shown in Figure 17. The un-irradiated control group gained and average of 3.2%
during the study, as compared with a mean loss of 12.1 % in the placebo group.
In the
groups receiving eritoran at 10 mg/kg, a mean weight loss of 7.8% was seen in
the
group treated on days 0-10 as compared with a net loss of 2.2% in the group
treated on
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days 0 to 3, a net loss of 7.3% in the group treated on days 3 to 6, and a net
loss of
8.9% for the group treated on days 6 to 9. To determine whether the
differences
observed in weight change were significant, a One-Way ANOVA on the mean Area
Under the Curve (AUC) data was performed. The results of this analysis are
shown in
Figure 18. Three groups receiving radiation were significantly different from
the un-
irradiated controls, the placebo group (P<0.001), the group treated with
eritoran from
day 0 until day 10 (P=0.014), and the group treated with eritoran from day 6
to day 9
(P=0.025). The groups treated with eritoran from day 0 until day 3 or from day
3 to
day 6 were not significantly different than the un-irradiated controls.
However, the
group treated with eritoran from day 0 until day 3 had significantly less
weight loss
than the placebo controls (P=0.030). The weight data was reanalyzed with the
data
from all animals dying during the study removed. The results of this analysis
are
shown in Figures 19 and 20. There was little change in the results of the One-
Way
ANOVA analysis, except that the group treated with eritoran from day 0 until
day 9
was not significantly different from the un-irradiated controls in this
analysis.
5.3 Tongue Histology
Each tongue was processed for routine hematoxylin and eosin histology and
slides were reviewed in a blinded manner. A total of 57 samples were
evaluated, and
of these, 9 were in the un-irradiated control group, 9 were in the placebo
group, 11
were in the group treated with eritoran at 10 mg/kg from day 0 to day 9, 11
were in the
group treated with eritoran at 10 mg/kg from day 0 to day 3, 9 were in the
group
treated with eritoran at 10 mg/kg from day 3 to day 6, and 9 were in the group
treated
with eritoran at 10 mg/kg from day 6 to day 9. Three sections from each sample
were
evaluated for the following parameters: epitheial score, connective tissue
score,
inflammation score, mitoses per 10 high power fields (hpf), percent
ulceration,
number of inflammatory cells per 10 hpf (percent neutrophils, lymphocytes, and
monocytes/macrophage), the number of small, medium, and large blood vessels
per 10
hpf, and the number of mast cells per 10 hpf.
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5.3.1 Epitlzelial Score
Epithelial histology was scored on a 4 point 0-3 scale as outlined in section
4.7.1. These scores are shown in Figure 21. The un-irradiated animals all had
scores
of 0. The placebo control group had a mean score of 1.1, as did the group
treated with
eritoran at 10 mg/kg from day 0 to day 9. The group treated with eritoran at
10 mg/kg
from day 0 to day 3 had a mean score of 0.45. The group treated with eritoran
at 10
mg/kg from day 3 to day 6 had a mean score of 0.89. The group treated with
eritoran
at 10 mg/kg from day 6 to day 9 had a mean score of 0.75.
5.3.2 Connective Tissue Score
Connective histology was scored on a 4 point 0-3 scale as outlined in section
4.7.1. These scores are shown in Figure 22. The un-irradiated animals all had-
scores
of 0. The placebo control group had a mean score of 0.4, the group treated
with
eritoran at 10 mg/kg from day 0 to day 9 had a mean score of 0.6. The group
treated
with eritoran at 10 mg/kg from day 0 to day 3 had a mean score of 0.4. The
group
treated with eritoran at 10 mg/kg from day 3 to day 6 had a mean score of 0.6.
The
group treated with eritoran at 10 mg/kg from day 6 to day 9 had a mean score
of 0.8.
5.3.3 Inflammation Score
Inflammation was scored on a 4 point 0-3 scale as outlined in section 4.7.1.
These scores are shown in Figure 23. The un-irradiated a.iiimals all had
scores of 0.
The placebo control group had a mean score of 0.4, the group treated with
eritoran at
10 mg/kg from day 0 to day 9 had a mean score of 0.5. The group treated with
eritoran at 10 mg/kg from day 0 to day 3 had a mean score of 0.4. The group
treated
with eritoran at 10 mg/kg from day 3 to day 6 had a mean score of 0.6. The
group
treated with eritoran at 10 mg/kg from day 6 to day 9 had a mean score of 0.8.
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5.3.4 Number of Mitoses
The number of mitoses was counted in 10 high power fields (hpf). These data
are shown in Figure 24. The un-irradiated animals had an average of 1.2
mitoses per
hpf. The placebo control group had an average of 3.9 mitoses per 10 hpf. The
5 group treated with eritoran at 10 mg/kg from day 0 to day 9 had an average
of 2.3
mitoses per 10 hpf. The group treated with eritoran at 10 mg/kg from day 0 to
day 3
had an average of 1.5 mitoses per 10 hpf. The group treated with eritoran at
10 mg/kg
from day 3 to day 6 had an average of 1.6 mitoses per 10 hpf. The group
treated with
eritoran at 10 mg/kg from day 6 to day 9 had an average of 1.5 mitoses per 10
hpf.
5.3.5 Percent Ulceration
The percentage ulceration was estimated for each sample. These data are
shown in Figure 25. The un-irradiated animals had no ulceration. The placebo
control group had mean ulceration of 13.3%. The group treated with eritoran at
10
mg/kg from day 0 to day 9 had mean ulceration of 13.2%. The group treated with
eritoran at 10 mg/kg from day 0 to day 3 had mean ulceration of 2.7%. The
group
treated with eritoran at 10 mg/kg from day 3 to day 6 had mean ulceration of
16.7%.
The group treated with eritoran at 10 mg/kg from day 6 to day 9 had mean
ulceration
of 10.0%.
5.3. 6 Inflamnzatofy Cell Infiltrates
The inflamnzatory cell infiltrate present in each sample was enumerated by
counting the total number of inflammatory cells per 10 hpf, and evaluated for
cell type
by estimating the percentage of cells within the infiltrate that were
neutrophils,
lymphocytes, or monocytes/macrophage. The numbers of inflammatory cell data
are
shown in Figure 26, the percent neutrophils in Figure 27, the percent
lymphocytes in
Figure 28, and the percent monocytes/macrophage in Figure 29. The un-
irradiated
animals had an average of 9.3 cells per 10 hpf, with an average composition of
98.9%
lymphocytes and 1.1 % monocytes/macrophage, with no neutrophils seen. The
placebo control group had an average of 44.9 cells per 10 hpf, with an average
composition of 10.6% neutrophils, 86.7% lymphocytes, and 2.8%
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monocytes/macrophage. The group treated with eritoran at 10 mg/kg from day 0
to
day 9 had an average of 43.6 cells per 10 hpf, with an average composition of
13.6%
neutrophils, 82.7% lymphocytes, and 4.5% monocytes/macrophage. The group
treated with eritoran at 10 mg/kg from day 0 to day 3 had an average of 33.3
cells per
10 hpf, with an average composition of 6.4% neutrophils, 93.2% lymphocytes,
and
0.5% monocytes/macrophage. The group treated with eritoran at 10 mg/kg from
day 3
to day 6 had an average of 31.5 cells per 10 hpf, with an average composition
of 7.2%
neutrophils, 91.1 % lymphocytes, and 1.1 % monocytes/macrophage. The group
treated with eritoran at 10 mg/kg from day 6 to day 9 had an average of 52.1
cells per
10 hpf, with an average conzposition of 8% neutrophils, 91.0% lymphocytes, and
1.0% monocytes/macrophage.
5.3.7 Blood Vessels
The number of blood vessels present in each sample was quantified by
counting the total number of blood vessels in 10 hpf, and evaluated for vessel
size by
counting the number of small, medium, and large vessels in this sample. These
data
are shown in Figures 30-32. The un-irradiated animals had an average of 5.6
blood
vessels per 10 hpf, with an average composition of 63.3 1o small, 20.7%
medium, and
16.0% large vessels seen. The placebo control group had an average of 8.8
blood
vessels per 10 hpf, with an average composition of 63.9% small, 22.3% medium,
and
13.9% large vessels. The group treated with eritoran at 10 mg/kg from day 0 to
day 9
had an average of 9.4 blood vessels per 10 hpf, with an average composition of
74.6%
small, 16.1% medium, and 9.3% large vessels seen. The group treated with
eritoran at
10 mg/kg from day 0 to day 3 had an average of 8.2 blood vessels per 10 hpf,
with an
average composition of 72.5% small, 14.9% medium, and 12.6% large vessels. The
group treated with eritoran at 10 mg/kg from day 3 to day 6 had an average of
7.0
blood vessels per 10 hpf, with an average composition of 67.9% small, 20.5%
medium, and 11.6% large vessels. The group treated with eritoran at 10 mg/kg
from
day 6 to day 9 had an average of 7.5 blood vessels per 10 hpf, with an average
composition of 72.1% small, 17.3% medium, and 10.6% large vessels.
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5.3.8. Mast Cells
The number of mast cells present in each sample was determined by counting
the number cells per 10 hpf. These data are shown in Figure 33. The un-
irradiated
animals had 23.7 mast cells per 10 hpf. The placebo control group had 26 mast
cells
per 10 hpf. The group treated with eritoran at 10 mg/kg from day 0 to day 9
had 18.4
mast cells per 10 hpf. The group treated with eritoran at 10 mg/kg from day 0
to day 3
had 24.4 mast cells per 10 hpf. The group treated with eritoran at 10 mg/kg
from day
3 to day 6 had 24.2 mast cells per 10 hpf. The group treated with eritoran at
10 mg/kg
from day 6 to day 9 had 24.5 mast cells per 10 hpf.
5.4 Serum Cytokine Levels
Serum levels of TNF-a, IL-6, and SAA were measured using commercially
available ELISA kits.
5.4.1 Serum TNF-a Levels
The un-irradiated animals had serum TNF-a levels of 43.0 pg/mL. The
placebo control group had mean serum TNF-a levels of 63.8 pg/mL. The group
treated with eritoran at 10 mg/kg from day 0 to day 9 had mean serum TNF-a
levels of
62.0 pg/mL. The group treated with eritoran at 10 mg/kg from day 0 to day 3
had
mean serum TNF-a levels of 20.3 pg/mL. The group treated with eritoran at 10
mg/kg from day 3 to day 6 had mean serum TNF-a levels of 40.2 pg/mL. The group
treated with eritoran at 10 mg/kg from day 6 to day 9 had mean serum TNF-a
levels of
119.1 pg/mL. These data are shown in Figure 34.
5.4.2 Serum IL-6 Levels
The un-irradiated animals had serum IL-6 levels of 48.7 pg/mL. The placebo
control group had mean serum IL-6 levels of 154.2 pg/mL. The group treated
with
eritoran at 10 mg/kg from day 0 to day 9 had mean serum IL-6 levels of 85.6
pg/mL.
The group treated with eritoran at 10 mg/kg from day 0 to day 3 had mean serum
IL-6
levels of 19.7 pg/mL. The group treated with eritoran at 10 mg/kg from day 3
to day 6
had mean serum IL-6 levels of 50.4 pg/mL. The group treated with eritoran at
10
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mg/kg from day 6 to day 9 had mean serum IL-6 levels of 119.3 pg/mL. These
data
are shown in Figure 35.
5.4.3 Serum SAA Levels
The un-irradiated animals had serum SAA levels of 597 g/mL. The placebo
control group had mean serum SAA levels of 427 g/mL. The group treated with
eritoran at 10 mg/kg from day 0 to day 9 had mean serum SAA levels of 344
g/mL.
The group treated with eritoran at 10 mg/kg from day 0 to day 3 had mean serum
SAA
levels of 279 g/mL. The group treated with eritoran at 10 mg/kg from day 3 to
day 6
had mean serum SAA levels of 475 g/mL. The group treated with eritoran at 10
mg/kg from day 6 to day 9 had mean serum SAA levels of 652 g/mL. These data
are
shown in Figure 36.
6. CONCLUSIONS
1. There was no evidence of toxicity with eritoran in the mortality or weight
loss data from this study. As with previous studies, mortality was high, but
evenly
distributed across groups.
2. Mice treated with eritoran on days 0-3 showed a significant improvement in
weight loss relative to the placebo treated control group.
3. The levels of oral mucositis observed in the placebo treated control mice
were lower than anticipated, and made it difficult to assess the impact of
eritoran on
the levels of oral mucositis seen.
4. Possibly due to the relatively low levels of mucositis seen in the placebo
control group, little effect was seen in the group treated with eritoran at 10
mg/kg
from day 0 to day 9, in contrast to previous observations of efficacy with
this
treatment protocol.
5. Among the groups receiving radiation, the group treated with eritoran on
days 0-3 had the lowest epithelial score, connective tissue score, and percent
ulceration, indicating that it had suffered less damage than other groups.
This group
also had the lowest inflammation score and was second lowest in the number of
inflammatory cells and mitoses.
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6. Among the groups receiving radiation, the group treated with eritoran on
days 0-3 had the lowest serum levels of TNF-a, IL-6, and SAA, showing the
efficacy
of this regimen in reducing inflammatory responses.
What is claimed is:
