U.S. patent application number 12/775574 was filed with the patent office on 2010-11-04 for methods of reducing the severity of mucositis.
This patent application is currently assigned to Eisai R&D Management Co., Ltd.. Invention is credited to Tetsu Kawano, Seiichi Kobayashi, Hiroshi Shirota, Minghuang Zhang.
Application Number | 20100279978 12/775574 |
Document ID | / |
Family ID | 46325497 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279978 |
Kind Code |
A1 |
Kawano; Tetsu ; et
al. |
November 4, 2010 |
METHODS OF REDUCING THE SEVERITY OF MUCOSITIS
Abstract
The invention provides methods of reducing the severity of
mucositis, involving administration of a toll-like receptor 4
antagonist.
Inventors: |
Kawano; Tetsu; (Kobe,
JP) ; Kobayashi; Seiichi; (Belmont, MA) ;
Zhang; Minghuang; (Windham, NH) ; Shirota;
Hiroshi; (Belmont, MA) |
Correspondence
Address: |
McCarter & English, LLP & Eisai Co.
265 Franklin Street
Boston
MA
02110
US
|
Assignee: |
Eisai R&D Management Co.,
Ltd.
Tokyo
JP
|
Family ID: |
46325497 |
Appl. No.: |
12/775574 |
Filed: |
May 7, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11434019 |
May 15, 2006 |
7727974 |
|
|
12775574 |
|
|
|
|
10486455 |
Jul 26, 2004 |
|
|
|
11434019 |
|
|
|
|
60680733 |
May 13, 2005 |
|
|
|
60311325 |
Aug 10, 2001 |
|
|
|
Current U.S.
Class: |
514/53 |
Current CPC
Class: |
A61K 31/739 20130101;
A61K 31/704 20130101; A61K 31/35 20130101; A61P 29/00 20180101;
A61K 9/0019 20130101; A61P 31/00 20180101; A61K 31/663 20130101;
A61K 47/26 20130101 |
Class at
Publication: |
514/53 |
International
Class: |
A61K 31/7016 20060101
A61K031/7016; A61P 29/00 20060101 A61P029/00; A61P 31/00 20060101
A61P031/00 |
Claims
1. A method of reducing the severity of mucositis in a patient, the
method comprising the step of administering to the patient a
composition comprising a compound that blocks activation of
toll-like receptor 4.
2. The method of claim 1, wherein the compound is a lipid A
analog.
3. The method of claim 2, wherein the lipid A analog is within the
formula: ##STR00012## where R.sup.1 is selected from the group
consisting of: ##STR00013## where each J, K, and Q, independently,
is straight or branched C1 to C15 alkyl; L is O, NH, or CH.sub.2; M
is O or NH; and G is NH, O, S, SO, or SO.sub.2; R.sup.2 is straight
or branched C5 to C15 alkyl; R.sup.3 is selected from the group
consisting of straight or branched C5 to C18 alkyl, ##STR00014##
where E is NH, O, S, SO, or SO.sub.2; each A, B, and D,
independently, is straight or branched C1 to C15 alkyl; R.sup.4 is
selected from the group consisting of straight or branched C4 to
C20 alkyl, and ##STR00015## where each U and V, independently, is
straight or branched C2 to C15 alkyl and W is hydrogen or straight
or branched C1 to C5 alkyl; R.sub.A is R.sup.5 or
R.sup.5--O--CH.sub.2--, R.sup.5 being selected from the group
consisting of hydrogen, J', -J'-OH, -J'-O--K', -J'-O--K'--OH, and
-J'-O--PO(OH).sub.2, where each J' and K', independently, is
straight or branched C1 to C5 alkyl; R.sup.6 is selected from the
group consisting of hydroxy, halogen, C1 to C5 alkoxy and C1 to C5
acyloxy; A.sup.1 and A.sup.2, independently, are selected from the
group consisting of ##STR00016## where Z is straight or branched C1
to C10 alkyl; or a pharmaceutically acceptable salt or phosphate
ester thereof.
4. The method of claim 3, wherein the lipid A analog is of the
structure: ##STR00017## or a pharmaceutically acceptable salt or
phosphate ester thereof.
5. The method of claim 4, wherein the lipid A analog is of the
structure: ##STR00018## or a pharmaceutically acceptable salt or
phosphate ester thereof.
6. The method of claim 1, wherein the mucositis is oral
mucositis.
7. The method of claim 1, wherein the mucositis is of the
gastrointestinal tract.
8. The method of claim 1, wherein the patient has mucositis.
9. The method of claim 1, wherein the patient does not have, but is
at risk of developing, mucositis.
10. The method of claim 9, wherein development of mucositis is
inhibited in the patient by administration of the composition.
11. The method of claim 10, wherein development of mucositis is
prevented in the patient by administration of the composition.
12. The method of claim 1, wherein the patient is a cancer
patient.
13. The method of claim 1, wherein the patient has recently been,
will shortly be, or is currently subject to treatment with head or
neck irradiation, or stem cell or bone marrow transplantation.
14. The method of claim 1, wherein said administration step occurs
prior to, concurrently with, or after a treatment that places the
patient at risk of developing mucositis, or a combination
thereof.
15. The method of claim 14, wherein said administration step occurs
prior to a treatment that places the patient at risk of developing
mucositis.
16. The method of claim 14, wherein said administration step occurs
concurrently with a treatment that places the patient at risk of
developing mucositis.
17. The method of claim 14, wherein said administration step occurs
after treatment that places the patient at risk of developing
mucositis.
18. The method of claim 14, wherein said administration step occurs
concurrently with a treatment that places the patient at risk of
developing mucositis, further comprising a step of administering
the composition at least once during days 0-3 after the treatment
that places the patient at risk of developing mucositis.
19. The method of claim 14, wherein the treatment that places the
patient at risk of developing mucositis comprises radiation
therapy.
20. The method of claim 14, wherein the treatment that places the
patient at risk of developing mucositis comprises chemotherapy.
21. The method of claim 1, wherein the composition is administered
to the patient topically.
22. The method of claim 1, wherein the composition is administered
to the patient by intravenous infusion.
23. The method of claim 1, further comprising the step of
administering antimicrobial therapy to the patient.
24. The method of claim 23, wherein the antimicrobial therapy is
antibiotic therapy.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/486,455, filed Jul. 26, 2004, which is a filing under 35 U.S.C.
.sctn.371 of PCT/US02/25452, filed Aug. 12, 2002, which claims
priority under 35 U.S.C. .sctn.119(e) from U.S. Ser. No.
60/311,325, filed Aug. 10, 2001. This application also claims
priority under 35 U.S.C. .sctn.119(e) from U.S. Ser. No.
60/680,733, filed May 13, 2005. The contents of each of the prior
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to methods for reducing the severity
of mucositis.
[0003] 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.
[0004] 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
preventing and treating mucositis is therefore important for
improving the care of cancer patients.
SUMMARY OF THE INVENTION
[0005] The invention provides methods of reducing the severity of
mucositis (e.g., 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:
##STR00001##
where R.sup.1 is selected from the group consisting of:
##STR00002##
where each J, K, and Q, independently, is straight or branched C1
to C15 alkyl; L is O, NH, or CH.sub.2; M is O or NH; and G is NH,
O, S, SO, or SO.sub.2; [0006] R.sup.2 is straight or branched C5 to
C15 alkyl; [0007] R.sup.3 is selected from the group consisting of
straight or branched C5 to C18 alkyl,
##STR00003##
[0007] where E is NH, O, S, SO, or SO.sub.2; each A, B, and D,
independently, is straight or branched C1 to C15 alkyl; [0008]
R.sup.4 is selected from the group consisting of straight or
branched C4 to C20 alkyl, and
##STR00004##
[0008] where each U and V, independently, is straight or branched
C2 to C15 alkyl and W is hydrogen or straight or branched C1 to C5
alkyl; [0009] R.sub.A is R.sup.5 or R.sup.5--O--CH.sub.2--, R.sup.5
being selected from the group consisting of hydrogen, J', -J'-OH,
-J'-O--K', -J'-O--K'--OH, and -J'-O--PO(OH).sub.2, where each J'
and K', independently, is straight or branched C1 to C5 alkyl;
[0010] R.sup.6 is selected from the group consisting of hydroxy,
halogen, C1 to C5 alkoxy and C1 to C5 acyloxy; [0011] A.sup.1 and
A.sup.2, independently, are selected from the group consisting
of
##STR00005##
[0011] where Z is straight or branched C1 to C10 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.sup.1 or A.sup.2 can be substituted to form a phosphate
ester.
[0012] An example of a Lipid A analog that can be included in the
compositions of the invention is a compound having the following
structure:
##STR00006##
[0013] or a pharmaceutically acceptable salt or phosphate ester
thereof.
[0014] In a more specific example, the compound is of the following
structure:
##STR00007##
or a pharmaceutically acceptable salt or phosphate ester
thereof.
[0015] Patients that can be treated according to the invention
include those who have mucositis (e.g., oral or gastrointestinal
mucositis). In addition, patients who do not have, but are at risk
of developing, mucositis (e.g., 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.
[0016] Examples of treatments that may cause or place a patient at
risk of developing mucositis (e.g., 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.
[0017] 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 mucositis (e.g., 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.
[0018] 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,
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 further below.
[0019] Further, the invention includes compositions including the
compounds 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.
[0020] 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.
[0021] Other features and advantages of the invention will be
apparent from the following detailed description, the drawings, and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 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.
[0023] FIG. 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).
[0024] FIG. 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.
[0025] FIG. 4 is a graph showing the mean serum TNF-.alpha.
concentration of snout irradiation-treated C3H/HeOuJ and C3H/HeJ
mice measured by ELISA analysis at the indicated time points.
[0026] FIG. 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.
[0027] FIG. 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.
[0028] FIG. 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.
[0029] FIG. 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.
[0030] FIG. 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.
[0031] FIG. 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.
[0032] FIG. 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.
[0033] FIG. 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 FIG. 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).
[0034] FIG. 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.
[0035] FIG. 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.
[0036] FIG. 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.
[0037] FIG. 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.
[0038] FIG. 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 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.
[0039] FIG. 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 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=0.030).
[0040] FIG. 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.
[0041] FIG. 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).
[0042] FIG. 21 is a graph showing mean epithelial scores and
standard errors of the mean for each of the indicated groups.
[0043] FIG. 22 is a graph showing mean connective tissue scores and
standard errors of the mean for each of the indicated groups.
[0044] FIG. 23 is a graph showing mean inflammation scores and
standard errors of the mean for each of the indicated groups.
[0045] FIG. 24 is a graph showing mean number of mitoses per 10 hpf
and standard errors of the means for each of the indicated
groups.
[0046] FIG. 25 is a graph showing the mean percent ulceration and
standard error of the mean for each of the indicated groups.
[0047] FIG. 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.
[0048] FIG. 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.
[0049] FIG. 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.
[0050] FIG. 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.
[0051] FIG. 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.
[0052] FIG. 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.
[0053] FIG. 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.
[0054] FIG. 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.
[0055] FIG. 34 is a graph showing serum TNF-.alpha. levels measured
using an ELISA assay and the mean and standard error of the mean
for each of the indicated groups.
[0056] FIG. 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.
[0057] FIG. 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.
DETAILED DESCRIPTION
[0058] The present invention provides methods of reducing the
severity of mucositis (e.g., 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.
[0059] 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.
[0060] 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:
##STR00008##
or a pharmaceutically acceptable salt or phosphate ester
thereof.
[0061] In a more specific example, the compound is of the following
structure:
##STR00009##
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. Pat. No.
5,935,938.
[0062] Other examples of compounds that can be used in the
invention include the following:
##STR00010## ##STR00011##
and a pharmaceutically acceptable salt or phosphate ester
thereof.
[0063] Additional TLR4 antagonists that can be used in the
invention include, for example, compound B531 (U.S. Pat. No.
5,530,113), as well as other compounds described in the following
patents: U.S. Pat. No. 5,935,938; U.S. Pat. No. 5,612,476; U.S.
Pat. No. 5,756,718; U.S. Pat. No. 5,843,918; U.S. Pat. No.
5,750,664; U.S. Pat. No. 6,235,724; U.S. Pat. No. 6,184,366; and
U.S. Pat. No. 5,681,824. Methods for making these compounds are
also described in these documents. Additional methods for making
such drugs are described, for example, in WO 02/94019.
[0064] 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 mucositis (e.g., 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 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.
[0065] 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 chemotherapy 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, bleomycin, 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.
[0066] 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.
[0067] 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 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.
[0068] 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. Pat. 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
further below.
[0069] 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 Remington's Pharmaceutical Sciences
(18.sup.th edition), ed. A. Gennaro, 1990, Mack Publishing Company,
Easton, Pa. (also see, e.g., M. J. Rathbone, ed., Oral Mucosal Drug
Delivery, Drugs and the Pharmaceutical 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.
[0070] 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) .mu.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.
[0071] When administering the compound eritoran by intravenous
infusion, it is preferable to use devices and equipment (e.g.,
catheters, such as central or peripheral venous catheters, tubing,
drip chambers, flashback bulbs, injection Y sites, stopcocks, and
infusion 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.
[0072] 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.
[0073] The methods of the invention can be used alone or in
conjunction with other approaches to reducing the severity of
mucositis. For example, 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.
[0074] 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, 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; Kepivance.TM.; 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).
[0075] The present invention is based, in part, on the following
experimental results.
Example I
1. Introduction
1.1 Rationale
[0076] 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 a
localized acute radiation to the snout. The mechanistic basis for
these differences is not understood.
1.2 Acute Snout Radiation Model
[0077] 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
[0078] 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-.alpha. and IL-6.
2.2 Study Summary
[0079] 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 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.
TABLE-US-00001 TABLE 1 Histological and cytokine comparison of the
effects of ionizing radiation of the oral mucosa of C3H/HeJ and
C3H/HeOuJ mice Hour/Day 0 2 H 6 H 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Randomize animals X Weigh, Record survival X X X X X X X X X X X X
X X X Irradiate all animals 30 Gy to snout X Sacrifice 4
animals/group (1 and 2) X X X X X X X dissect oral mucosa Sacrifice
4 animals/group (3 and 4) X dissect oral mucosa Obtain serum from
each animal X X X X X X X
3. Study Design
[0080] 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.
TABLE-US-00002 TABLE 2 Animal allocation by experimental group.
Mouse Radiation Group Strain N 30 Gy 1 C3H/HeOuJ 28 YES WT 2
C3H/HeJ 28 YES Mutant 3 C3H/HeOuJ 4 NO WT 4 C3H/HeJ 4 NO Mutant
[0081] Every day for the period of the study (day 0 to day 14),
each animal was weighed. Animals in groups 1 and 2 received a
single dose of 30 Gy radiation 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-.alpha. and IL-6 using a standard ELISA assay.
4. Material and Methods
4.1 Animals
[0082] 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 approximately 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 Housing
[0083] The study was performed in animal rooms provided with
filtered air at a temperature of 70.degree. F.+/-5.degree. 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.RTM. 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 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
[0084] Animals were fed with a Labdiet.RTM. 5001 chow and water was
provided ad libitum.
4.4 Animal Randomization and Allocations
[0085] 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
[0086] 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
[0087] 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.
[0088] 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 time points were 2 hours, 6
hours, 24 hours, 3 days, 6 days, 10 days, and 14 days after
radiation.
[0089] 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.
[0090] 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-.alpha. and IL-6.
4.6.2 Cytokine ELISA
[0091] Enzyme linked immunosorbent assays (ELISAs) were performed
for cytokines TNF-.alpha. 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.degree. C. If insufficient
serum had been collected to run both IL-6 and TNF-.alpha., samples
were diluted 1:2 or 1:4, and run in duplicate in both assays. All
assays were performed using 50 .mu.L of sample per well.
4.6.3 Histology
[0092] 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) and reviewed by a board certified pathologist.
4.7 Assessment of Results
[0093] 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 Survival
[0094] 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 (FIGS. 1 and 2)
[0095] The mean percentage weight change for each group is shown in
FIG. 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 FIG.
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
[0096] Serum levels of cytokines IL-6 and TNF-.alpha. were
evaluated by ELISA.
5.3.1 Serum IL-6 Concentrations
[0097] 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 the day 10
time-point, when serum IL-6 concentrations increased to 69.4 pg/mL.
These data are shown in FIG. 3.
5.3.2 Serum TNF-.alpha. Concentrations
[0098] The mean serum TNF-.alpha. concentration in un-irradiated
C3H/HeOuJ mice was 48.0 pg/mL. There were 2 peaks in serum
TNF-.alpha. 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-.alpha. 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-.alpha.
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
FIG. 4.
5.4 Tongue Histology
[0099] 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
[0100] The epithelium and connective tissue regions of each sample
were each given separate scores. The scores for the epithelium are
shown in FIG. 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 is shown in FIG. 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 Inflammation
[0101] 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 FIG. 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 almost
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
[0102] 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 FIG. 8. The
number of mitotic figures counted in the epithelial 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
[0103] 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 FIG.
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 C3H/HeOuJ 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 FIG. 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.
6. Conclusions
[0104] 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).
[0105] 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 TNF-.alpha. being seen on day 6 post
irradiation.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
Example II
1. Introduction
1.1 Rationale
[0110] 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
[0111] 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
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
[0112] 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 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 Summary
[0113] 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
[0114] 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 animals 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.
TABLE-US-00003 TABLE 2 Allocation by experimental group. Number of
Group animals Strain Treatment Radiation Sac points 1 16 male
C3H/HeOuJ placebo IV bid 30 Gy to snout 8 on day 6, 8 on day 10 2
16 male C3H/HeOuJ eritoran 1 mg/kg IV bid 30 Gy to snout 8 on day
6, 8 on day 10 3 16 male C3H/HeOuJ eritoran 10 mg/kg IV 30 Gy to
snout 8 on day 6, bid 8 on day 10 4 6 male C3H/HeOuJ placebo IV bid
none 6 on day 10
4. Material and Methods
4.1 Animals
[0115] 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 Housing
[0116] The study was performed in animal rooms as described above
in section 4.2 of Example I.
4.3 Diet
[0117] Animals were fed with Labdiet.RTM. 5061 sterile irradiated
rodent chow and water was provided ad libitum.
4.4 Animal Randomization and Allocations
[0118] 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
[0119] 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
[0120] 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
[0121] 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 (FIGS. 11 and 12)
[0122] The mean percentage weight gain for each group for each day
of the study is shown in FIG. 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 FIG. 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).
5.3 Tongue Histology
[0123] 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).
[0124] 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 samples 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.
[0125] 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 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
[0126] 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 FIG. 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
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 FIG. 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 minimum 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 FIG. 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 shown in FIG. 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
[0127] 1. Significant mortality was seen during this study, but
this excess mortality was not associated with any one treatment
group.
[0128] 2. No statistically significant differences in weight gain
were seen between the three irradiated treatment groups.
[0129] 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.
[0130] 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.
Example III
1. Introduction
1.1 Rationale
[0131] 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
[0132] 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
[0133] The objective of this study was to examine the effect of
scheduling of eritoran, administered intravenously, 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. These samples were used for the measurement of serum
Tumor Necrosis Factor (TNF-.alpha.), Interleukin-6 (IL-6), and
Serum Amyloid A (SAA) levels.
2.2 Study Summary
[0134] 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
[0135] 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.
TABLE-US-00004 TABLE 4 Allocation by experimental group Number of
Dose Group Animals Treatment Eritoran Placebo Volume 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 10 mg/kg bid 4 10 Male
eritoran days 0-3 Days 0-3 Days 4-9 0.1 mL 10 mg/kg bid 5 10 Male
eritoran days 3-6 Days 3-6 Days 0-2 & 0.1 mL 10 mg/kg 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
serum was stored at -80.degree. C.
4. Material and Methods
4.1 Animals
[0136] 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 Housing.
[0137] The study was performed in animal rooms as described above
in section 4.2 of Example I.
4.3 Diet
[0138] Animals were fed with Labdiet.RTM. 5061 sterile irradiated
rodent chow and water was provided ad libitum.
4.4 Animal Randomization and Allocations
[0139] 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
[0140] 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
[0141] Histological samples 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
[0142] Enzyme linked immunosorbent assays (ELISAs) were performed
for cytokines TNF-.alpha. 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 manufacturer's instructions. All determinations
were made in duplicate on serum samples stored at -80.degree. C.
Samples were run in duplicate in all three assays, and if
insufficient serum had been collected to run IL-6, SAA, and
TNF-.alpha. assays, samples were diluted 1:4. All assays were
performed using 50 .mu.L of sample per well.
4.7 Assessment of Results
[0143] 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
[0144] 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 Weights (FIGS. 17, 18, 19, and 20)
[0145] The mean percentage weight gain for each group for each day
of the study is shown in FIG. 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 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 FIG. 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
FIGS. 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
[0146] 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.
5.3.1 Epithelial Score
[0147] Epithelial histology was scored on a 4 point 0-3 scale as
outlined in section 4.7.1. These scores are shown in FIG. 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
[0148] Connective histology was scored on a 4 point 0-3 scale as
outlined in section 4.7.1. These scores are shown in FIG. 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
[0149] Inflammation was scored on a 4 point 0-3 scale as outlined
in section 4.7.1. These scores are shown in FIG. 23. 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 0.5. 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.
5.3.4 Number of Mitoses
[0150] The number of mitoses was counted in 10 high power fields
(hpf). These data are shown in FIG. 24. The un-irradiated animals
had an average of 1.2 mitoses per 10 hpf. The placebo control group
had an average of 3.9 mitoses per 10 hpf. The 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
[0151] The percentage ulceration was estimated for each sample.
These data are shown in FIG. 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 Inflammatory Cell Infiltrates
[0152] The inflammatory 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 FIG. 26, the percent neutrophils in FIG. 27, the percent
lymphocytes in FIG. 28, and the percent monocytes/macrophage in
FIG. 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%
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 composition of 8% neutrophils,
91.0% lymphocytes, and 1.0% monocytes/macrophage.
5.3.7 Blood Vessels
[0153] 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
FIGS. 30-32. The un-irradiated animals had an average of 5.6 blood
vessels per 10 hpf, with an average composition of 63.3% 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.
5.3.8. Mast Cells
[0154] The number of mast cells present in each sample was
determined by counting the number cells per 10 hpf. These data are
shown in FIG. 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
[0155] Serum levels of TNF-.alpha., IL-6, and SAA were measured
using commercially available ELISA kits.
5.4.1 Serum TNF-.alpha. Levels
[0156] The un-irradiated animals had serum TNF-.alpha. levels of
43.0 pg/mL. The placebo control group had mean serum TNF-.alpha.
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-.alpha. 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-.alpha. 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-.alpha. 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-.alpha.
levels of 119.1 pg/mL. These data are shown in FIG. 34.
5.4.2 Serum IL-6 Levels
[0157] 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 mg/kg from day 6 to
day 9 had mean serum IL-6 levels of 119.3 pg/mL. These data are
shown in FIG. 35.
5.4.3 Serum SAA Levels
[0158] The un-irradiated animals had serum SAA levels of 597
.mu.g/mL. The placebo control group had mean serum SAA levels of
427 .mu.g/mL. The group treated with eritoran at 10 mg/kg from day
0 to day 9 had mean serum SAA levels of 344 .mu.g/mL. The group
treated with eritoran at 10 mg/kg from day 0 to day 3 had mean
serum SAA levels of 279 .mu.g/mL. The group treated with eritoran
at 10 mg/kg from day 3 to day 6 had mean serum SAA levels of 475
.mu.g/mL. The group treated with eritoran at 10 mg/kg from day 6 to
day 9 had mean serum SAA levels of 652 .mu.g/mL. These data are
shown in FIG. 36.
6. Conclusions
[0159] 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.
[0160] 2. Mice treated with eritoran on days 0-3 showed a
significant improvement in weight loss relative to the placebo
treated control group.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 6. Among the groups receiving radiation, the group treated
with eritoran on days 0-3 had the lowest serum levels of
TNF-.alpha., IL-6, and SAA, showing the efficacy of this regimen in
reducing inflammatory responses.
* * * * *