U.S. patent application number 13/013340 was filed with the patent office on 2011-05-19 for method of treating or preventing tissue deterioration, injury or damage due to disease of mucosa.
This patent application is currently assigned to SCICLONE PHARMACEUTICALS, INC.. Invention is credited to Israel RIOS, Cynthia TUTHILL.
Application Number | 20110117211 13/013340 |
Document ID | / |
Family ID | 39690681 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110117211 |
Kind Code |
A1 |
RIOS; Israel ; et
al. |
May 19, 2011 |
Method of Treating or Preventing Tissue Deterioration, Injury or
Damage Due to Disease of Mucosa
Abstract
An immunomodulatory compound is utilized to treat mucosa
disease.
Inventors: |
RIOS; Israel; (Menlo Park,
CA) ; TUTHILL; Cynthia; (Menlo Park, CA) |
Assignee: |
SCICLONE PHARMACEUTICALS,
INC.
Foster City
CA
|
Family ID: |
39690681 |
Appl. No.: |
13/013340 |
Filed: |
January 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12526585 |
Aug 10, 2009 |
7906486 |
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PCT/US08/01768 |
Feb 11, 2008 |
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13013340 |
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60900977 |
Feb 13, 2007 |
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Current U.S.
Class: |
424/649 ;
514/419 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
37/02 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61P
29/00 20180101; A61P 37/04 20180101; A61P 1/02 20180101; A61P 17/02
20180101; A61P 31/04 20180101; A61K 31/405 20130101 |
Class at
Publication: |
424/649 ;
514/419 |
International
Class: |
A61K 33/24 20060101
A61K033/24; A61K 31/405 20060101 A61K031/405; A61P 35/00 20060101
A61P035/00; A61P 43/00 20060101 A61P043/00 |
Claims
1-16. (canceled)
17. A method for reducing the amount of mucosal deterioration,
injury, or damage to at least one of oral and esophageal mucosal
tissues, the mucosal deterioration, injury, or damage resulting
from radiation treatment in a subject undergoing radiation therapy
for head and neck cancer, the method comprising: administering to
said subject an effective amount of a compound of the formula
##STR00003## or a pharmaceutically acceptable salt thereof,
wherein: n is 1 or 2; R is hydrogen or a C2-10 acyl or C1-6 alkyl;
and X is L-tryptophan or D-tryptophan.
18. The method of claim 17, wherein said compound is not
.gamma.-D-glutamyl-L-tryptophan (SCV-07) or a pharmaceutically
acceptable salt thereof.
19. The method of claim 17, wherein the oral and/or esophageal
mucosal deterioration, injury, or damage comprises ulcerative
lesions.
20. The method of claim 17, wherein said radiation is administered
at a cumulative dose of at least about 15 Gy.
21. The method of claim 17, wherein said radiation is administered
at a cumulative dose of at least about 40 Gy.
22. The method of claim 17, wherein said radiation is administered
at a cumulative dose of at least about 60 Gy.
23. The method of claim 17, wherein a chemotherapy is administered
to the subject.
24. The method of claim 23, wherein the chemotherapy is
cis-platin.
25. The method of claim 24, wherein said cis-platin is administered
at a dosage within a range of 0.1-50 mg/kg.
26. The method of claim 24, wherein said cis-platin is administered
at a dosage of about 5 mg/kg.
27. The method of claim 17 wherein said compound or a
pharmaceutically acceptable salt thereof is administered before
radiation exposure.
28. The method of claim 17 wherein said compound or a
pharmaceutically acceptable salt thereof is administered during
radiation exposure.
29. The method of claim 17 wherein said compound or a
pharmaceutically acceptable salt thereof is administered after
radiation exposure.
30. The method of claim 17 wherein said compound or a
pharmaceutically acceptable salt thereof is administered before
administration of 7-8 Gy of radiation.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of U.S. Provisional
Application No. 60/900,977, filed Feb. 13, 2007.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of mucosa disease
treatment.
[0004] 2. Description of the Background Art
[0005] Oral ulcerative mucositis is a common, painful,
dose-limiting toxicity of drug and radiation therapy for cancer.
The disorder is characterized by breakdown of the oral mucosa that
results in the formation of ulcerative lesions. In granulocytopenic
patients, the ulcerations that accompany mucositis are frequent
portals of entry for indigenous oral bacteria often leading to
sepsis or bacteremia. Mucositis occurs to some degree in more than
one third of patients receiving anti-neoplastic drug therapy. The
frequency and severity are significantly greater among patients who
are treated with induction therapy for leukemia or with many of the
conditioning regimens for bone marrow transplant. Among these
individuals, moderate to severe mucositis is not unusual in more
than three-quarters of patients. Moderate to severe mucositis
occurs in virtually all patients who receive radiation therapy for
tumors of the head and neck and typically begins with cumulative
exposures of 15 Gy and then worsens as total doses of 60 Gy or more
are reached.
[0006] Clinically mucositis progresses through three stages:
1. Inflammation accompanied by painful mucosal erythema, which can
respond to local anesthetics. 2. Painful ulceration with
pseudomembrane formation and, in the case of myelosuppressive
treatment, potentially life-threatening sepsis, requiring
antimicrobial therapy. Pain is often of such intensity as to
require parenteral narcotic analgesia. 3. Spontaneous healing,
occurring about 2-3 weeks after cessation of anti-neoplastic
therapy.
[0007] Standard therapy for mucositis is predominantly palliative,
including application of topical analgesics such as lidocaine
and/or systemic administration of narcotics and antibiotics.
Currently, there is only one approved treatment for oral mucositis,
Kepivance (Palifermin), which is only approved for the treatment of
oral mucositis in patients undergoing conditioning regimens prior
to hematopoetic stem cell transplantation for the treatment of
hematologic malignancies.
[0008] The complexity of mucositis as a biological process has only
been recently appreciated. It has been suggested that the condition
represents a sequential interaction of oral mucosal cells and
tissues, reactive oxygen species, pro-inflammatory cytokines,
mediators of apoptosis and local factors such as saliva and the
oral micro biota. While epithelial degeneration and breakdown
ultimately result in mucosal ulceration, it appears that the early
changes associated with radiation-induced mucosal toxicity occur
within the endothelium, and connective tissue of the submucosa.
Electron microscopic evaluation of mucosa within 1 week of
radiation shows damage to both endothelium and connective tissue,
but not epithelium. Such injury is likely mediated by free radical
formation. It appears that the overall mechanism for mucositis
development is similar for both radiation and chemotherapy.
[0009] There remains a need in the art for improvements in methods
for treating or preventing mucosa diseases.
SUMMARY OF THE INVENTION
[0010] In accordance with one aspect, a method of treatment for
treating, preventing, inhibiting or reducing tissue deterioration,
injury or damage due to disease of mucosa, or for restoring tissue
adversely affected by said disease, in a subject, comprises
administering to said target subject an effective amount of an
immunomodulator compound of formula A:
##STR00001##
[0011] In Formula A, n is 1 or 2, R is hydrogen, acyl, alkyl or a
peptide fragment, and X is an aromatic or heterocyclic amino acid
or a derivative thereof, wherein said immunomodulator compound is
administered to said target subject without administering said at
least one interferon to said target subject. Preferably, X is
L-tryptophan or D-tryptophan. The invention also relates to use of
a compound of formula A in preparation of a medicament for
treatment of mucosa disease.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] The present invention is applicable to mucosa diseases or
disease manifestations thereof. In preferred embodiments, the
mucosa disease results from radiation and/or chemotherapy of a
subject, preferably in a human patient. The radiation may be acute
or fractionated. The mucosa disease may result from a combination
of radiation and chemotherapy. In certain embodiments, the disease
is of oral and/or esophageal mucosa, e.g., mucositis and/or
ulcerative lesions.
[0013] In accordance with one embodiment, the present invention
relates to treatment of mucositis by administering an
immunomodulator compound to a mammalian subject, preferably a human
patient.
[0014] In certain embodiments, the immunomodulatory compounds of
the present invention are administered before, during and/or after
administration of radiation and/or a chemotherapeutic agent to a
patient. Radiation often is administered in multiple doses, and
mucosa disease occurs in virtually all patients who receive
radiation therapy for tumors of the head and neck, typically
beginning with cumulative exposures of 15 Gy radiation and then
worsening at total cumulative doses of 60 Gy radiation or more. In
preferred embodiments, an immunomodulator compound according to the
invention is administered before, during or after administration of
7-8 Gy (e.g., 7.5 Gy) radiation to a patient, 15 Gy, 40 Gy, 60 Gy,
or more radiation to patient.
[0015] The immunomodulator compounds of the invention also can be
administered for the treatment or prevention of mucosa disease
resulting from administration of chemotherapy agents, such as
cis-platin, e.g., administered at a dosage within a range of about
0.1-50 mg/kg, e.g., about 5 mg/kg.
[0016] Immunomodulator compounds used in accordance with the
present invention, comprise immunomodulators of Formula A:
##STR00002##
[0017] In Formula A, n is 1 or 2, R is hydrogen, acyl, alkyl or a
peptide fragment, and X is an aromatic or heterocyclic amino acid
or a derivative thereof. Preferably, X is L-tryptophan or
D-tryptophan. Appropriate derivatives of the aromatic or
heterocyclic amino acids for "X" are: amides, mono-or
di-(C.sub.1-C.sub.6) alklyl substituted amides, arylamides, and
(C.sub.1-C.sub.6) alkyl or aryl esters. Appropriate acyl or alkyl
moieties for "R" are: branched or unbranched alkyl groups of 1 to
about 6 carbons, acyl groups from 2 to about 10 carbon atoms, and
blocking groups such as carbobenzyloxy and t-butyloxycarbonyl.
Preferably the carbon of the CH group shown in Formula A has a
stereoconfiguration, when n is 2, that is different from the
stereoconfiguration of X.
[0018] Preferred embodiments utilize compounds such as
.gamma.-D-glutamyl-L-tryptophan, .gamma.-L-glutamyl-L-tryptophan,
.gamma.-L-glutamyl-N.sub.in-formyl-L-tryptophan,
N-methyl-.gamma.-L-glutamyl-L-tryptophan,
N-acetyl-.gamma.-L-glutamyl-L-tryptophan,
.gamma.-L-glutamyl-D-tryptophan, .beta.-L-aspartyl-L-tryptophan,
and .beta.-D-aspartyl-L-tryptophan. Particularly preferred
embodiments utilize .gamma.-D-glutamyl-L-tryptophan, sometimes
referred to as SCV-07. These compounds, methods for preparing these
compounds, pharmaceutically acceptable salts of these compounds and
pharmaceutical formulations thereof are disclosed in U.S. Pat. No.
5,916,878, incorporated herein by reference.
[0019] SCV-07, .gamma.-D-glutamyl-L-tryptophan, is a member of a
class of immunomodulatory drugs that possess .gamma.-glutamyl or
.beta.-aspartyl moieties, which was discovered by Russian
scientists and is being examined for efficacy in several
indications in the U.S. by SciClone Pharmaceuticals, Inc. SCV-07
possesses a number of immunomodulatory activities in vivo and in
vitro. SCV-07 increases Con-A-induced thymocyte and lymphocyte
proliferation, increases Con-A-induced interleukin-2 (IL-2)
production and IL-2 receptor expression by spleen lymphocytes, and
stimulates expression of Thy-1.2 on bone marrow cells. In vivo,
SCV-07 has a strong immunostimulatory effect on
5-FU-immune-suppressed animals and in a model of immunization with
sheep red blood cells.
[0020] The Formula A compounds may be administered as dosages in
the range of about 0.001-2000 mg, more preferably about 0.01-100
mg. Dosages may be administered one or more times per week,
preferably on a daily basis, with dosages administered one or more
times per day. Administration can be by any suitable method,
including orally, nasally, transdermally, sublingually, by
injection, periodic infusion, continuous infusion, and the like.
The dosages may be administered by intramuscular injection,
although other forms of injection and infusion may be utilized, and
other forms of administration such as oral or nasal inhalation or
oral ingestion may be employed.
[0021] Dosages may also be measured in micrograms per kilogram,
with dosages in the range of about 0.1-10,000 ug/kg, more
preferably within the range of about 1.0-1000 ug/kg.
[0022] Included are biologically active analogs having substituted,
deleted, elongated, replaced, or otherwise modified portions which
possess bioactivity substantially similar to that of SCV-07, e.g.,
an SCV-07 derived peptide having sufficient homology with SCV-07
such that it functions in substantially the same way with
substantially the same activity as SCV-07.
[0023] In some embodiments, the Formula A compound is present in a
pharmaceutically acceptable liquid carrier, such as water for
injection, saline in physiological concentrations, or similar, or
in tablet form with suitable dry carrier(s) and excipient(s).
[0024] Effective amounts of Formula A compound can be determined by
routine dose-titration experiments.
Example 1
A First Study of SCV-07 in the Treatment of Oral Mucositis Induced
by Acute Radiation in Hamsters
1. Objective
[0025] The objective of this study was to conduct a preliminary
evaluation of the efficacy of SCV-07 in the treatment of oral
mucositis using an acute radiation induced hamster model of the
disease. SCV-07 was given at doses of 1, 10 or 100 ug/kg once daily
by sub-cutaneous injection for 18 days, starting one day before
radiation and continuing until day 16 after radiation. No deaths
were observed in any of the treatment groups and there were no
statistically significant changes in growth rate, suggesting that
SCV-07 was well tolerated at these doses. Animals treated with
SCV-07 at 10 ug/kg on days -1 to 16 showed a statistically
significant reduction in mucositis scores on day 22 (P=0.024).
Animals treated with SCV-07 at 100 ug/kg on days -1 to 16 showed a
statistically significant reduction in mucositis scores on day 14
(P=0.025), in addition to a statistically significant reduction in
the number of animal days with a score of 3 or higher (P=0.029).
These data suggest that SCV-07 demonstrated a dose dependant
benefit on the severity and course of radiation induced
mucositis.
Acute Radiation Model
[0026] The acute radiation model in hamsters has proven to be an
accurate, efficient and cost-effective technique to provide a
preliminary evaluation of anti-mucositis compounds. The course of
mucositis in this model is well defined and results in peak scores
approximately 14-16 Days following radiation. The acute model has
little systemic toxicity, resulting in few hamster deaths, thus
permitting the use of smaller groups (N=7-8) for initial efficacy
studies. It has also been used to study specific mechanistic
elements in the pathogenesis of mucositis. Molecules that show
efficacy in the acute radiation model may be further evaluated in
the more complex models of fractionated radiation, chemotherapy, or
concomitant therapy.
[0027] In this study, an acute radiation dose of 40 Gy on day 0 was
administered. Clinically significant mucositis was observed on days
12 through 28.
2. Study Objective and Summary
Study Objective
[0028] The objective of this study was to evaluate the effect of
SCV-07, administered by sub-cutaneous injection on different
schedules between the day before radiation and the sixteenth day
after radiation, on the frequency, severity and duration of oral
mucositis induced by acute radiation.
Study Summary
[0029] Thirty-two (32) Syrian Golden Hamsters were given an acute
radiation dose of 40 Gy directed to their left buccal cheek pouch
on day 0. Test materials were given by sub-cutaneous injection once
daily. Dosing began one day before radiation (day -1) and continued
until day 16. Mucositis was evaluated clinically starting on day 6,
and continuing on alternate days until day 28.
3. Evaluation
Mucositis Evaluation
[0030] The grade of mucositis was scored, beginning day 6, and for
every second day thereafter, through and including day 28. The
effect on mucositis of each drug treatment compared to placebo was
assessed according to the following parameters:
[0031] The difference in the number of days hamsters in each group
have ulcerative (score .gtoreq.3) mucositis.
[0032] On each evaluation day, the number of animals with a blinded
mucositis score of .gtoreq.3 in each drug treatment group was
compared to the control group. Differences were compared on a
cumulative basis and statistical significance was determined by
chi-square analysis. Efficacy, in this analysis, is defined by a
significant reduction in the number of days that a group of animals
had ulcerations (scores .gtoreq.3) when compared to the control
group.
Rank Sum Differences in Daily Mucositis Scores
[0033] For each evaluation day the scores of the control group were
compared to those of the treated groups using non-parametric rank
sum analysis. Treatment success was considered as a statistically
significant lowering of scores in the treated group on 2 or more
days from day 6 to day 28.
Weights and Survival
[0034] All animals were weighed daily and their survival recorded,
in order to assess possible differences in animal weight among
treatment groups as an indication for mucositis severity and/or
possible toxicity resulting from the treatments.
4. Study Design
[0035] Thirty-two Syrian Golden Hamsters were divided into four (4)
groups of eight (8) animals each. All animals received a single
dose of acute radiation of 40 Gy directed to their left buccal
cheek pouch on day 0. This was accomplished by anesthetizing the
animals and everting the left buccal pouch, while protecting the
rest of the animals with a lead shield. Test materials were given
by sub-cutaneous injection once daily as detailed in Table 1.
Mucositis was evaluated clinically starting on Day 6, and
continuing on alternate days until day 28.
TABLE-US-00001 TABLE 1 Study Design Group Number of Treatment
Volume Number Animals Treatment Schedule* (mL) 1 8 males Vehicle
(PBS), sc, Day -1 to 16 0.1 mL/100 g qd 2 8 males SCV-07, sc, qd
Day -1 to 16 0.1 mL/100 g 1 .mu.g/kg, 3 8 males SCV-07, sc, qd Day
-1 to 16 0.1 mL/100 g 10 .mu.g/kg 4 8 males SCV-07, sc, qd Day -1
to 16 0.1 mL/100 g 100 .mu.g/kg *The dose on day 0 will be
performed 30 minutes prior to radiation
5. Material and Methods
Location of Study Performance
[0036] The study was performed at Biomodels AAALAC accredited
facility in Cambridge Mass. Approval for this study was obtained
from Biomodels IACUC.
Animals
[0037] Male LVG Syrian Golden Hamsters (Charles River
Laboratories), aged 5 to 6 weeks, with average body weight of 85.3
g at study commencement, were used. Animals were individually
numbered using an ear punch and housed in small groups of 8 animals
per cage. Animals were acclimatized prior to study commencement.
During this period of 5 days, the animals were observed daily in
order to reject animals that present in poor condition.
Housing
[0038] 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. Bedding was
changed a minimum of once per week. Cages, tops, bottles, etc. were
washed with a commercial detergent and allowed to air dry. 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 marked all cages. The temperature and relative
humidity was recorded during the study, and the records
retained.
Diet
[0039] Animals were fed with a Purina Labdiet.RTM. 5061 rodent diet
and water was provided ad libitum.
Animal Randomization and Allocations
[0040] Animals 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.
For more consistent identification, ear punch numbering was used
rather than tagging, since tags may become dislodged during the
course of the study. A cage card was used to identify each cage or
label marked with the study number (SCI-01), treatment group number
and animal numbers.
Sub-Cutaneous Dosing and Drug Application
[0041] The test compound, SCV-07 was provided as a powder and
dissolved in sterile PBS immediately prior to administration. Three
dilutions were prepared: 100 .mu.g/mL, 10 .mu.g/mL and 1 .mu.g/mL.
Drug was given in a volume of 0.1 mL per 100 g body weight, with
the appropriate dilution of SCV-07 for each group, using a
tuberculin syringe with a 27G needle. Injections were given
subcutaneously to the back or abdomen.
Mucositis Induction
[0042] Mucositis was induced using a standardized acute radiation
protocol. A single dose of radiation (40 Gy/dose) was administered
to all animals on day 0. Radiation was generated with a 160
kilovolt potential (15-ma) source at a focal distance of 30 cm,
hardened with a 0.35 mm Cu filtration system. Irradiation targeted
the left buccal pouch mucosa at a rate of 3.2 Gy/minute. Prior to
irradiation, animals were anesthetized with an intraperitoneal
injection of Ketamine (160 mg/kg) and Xylazine (8 mg/kg). The left
buccal pouch was everted, fixed and isolated using a lead shield to
protect all other parts of the hamster's body.
Mucositis Scoring
[0043] The mucositis score, weight change and survival were
measured throughout the study as described above. For the
evaluation of mucositis, the animals were anesthetized with an
inhalation anesthetic, and the left pouch everted. Mucositis was
scored visually by comparison to a validated photographic scale,
ranging from 0 for normal, to 5 for severe ulceration (clinical
scoring). In descriptive terms, this scale is defined as
follows:
TABLE-US-00002 TABLE 2 Mucositis Scoring. Score: Description: 0
Pouch completely healthy. No erythema or vasodilation. 1 Light to
severe erythema and vasodilation. No erosion of mucosa. 2 Severe
erythema and vasodilation. Erosion of superficial aspects of mucosa
leaving denuded areas. Decreased stippling of mucosa. 3 Formation
of off-white ulcers in one or more places. Ulcers may have a
yellow/gray due to pseudomembrane. Cumulative size of ulcers should
equal about 1/4 of the pouch. Severe erythema and vasodilation. 4
Cumulative seize of ulcers should equal about 1/2 of the pouch.
Loss of pliability. Severe erythema and vasodilation. 5 Virtually
all of pouch is ulcerated. Loss of pliability (pouch can only
partially be extracted from mouth).
[0044] A score of 1-2 is considered to represent a mild stage of
the disease, whereas a score of 3-5 is considered to indicate
moderate to severe mucositis. Following visual scoring, a
photograph was taken of each animal's mucosa using a standardized
technique. At the conclusion of the experiment, all films were
developed and the photographs randomly numbered. At least two
independent trained observers graded the photographs in blinded
fashion using the above-described scale (blinded scoring).
6. Results and Discussion
6.1 Survival
[0045] No deaths were observed during the course of this study.
6.2 Weight Change
[0046] The mean daily percent weight change data was evaluated. The
saline (PBS) treated control hamsters gained an average of 70.4% of
their starting weight during the study. Hamsters in the group
receiving SCV-07 at 1 ug/kg on days -1 to 16 gained an average of
68.4% of their starting weight during the study. Hamsters in the
group receiving SCV-07 at 10 ug/kg on days -1 to 16 gained an
average of 70.3% of their starting weight during the study.
Hamsters in the group receiving SCV-07 at 100 ug/kg on days -1 to
16 gained an average of 82.0% of their starting weight during the
study. The significance of these differences was evaluated by
calculating the area-under-the-curve (AUC) for the weight gain of
each animal, and then comparing the different treatment groups
using a One-Way ANOVA test. The results of this analysis indicated
that there were no significant differences between the different
treatment groups (P=0.191).
6.3 Mucositis (Tables 3 & 4)
[0047] Mean daily mucositis scores for each group were calculated.
In the saline treated control group, peak levels of mucositis were
seen on day 20, when the mean score reached 3.8. The group
receiving SCV-07 at 1 ug/kg from day -1 to 16 had a peak mean
mucositis score of 3.8 on day 16. The group receiving SCV-07 at 10
ug/kg from day -1 to 16 had a peak mean mucositis score of 3.7 on
day 16. The group receiving SCV-07 at 100 ug/kg from day -1 to 16
had a peak mean mucositis score of 3.4 on day 18. The significance
of the differences observed between the different treatment groups
was evaluated by calculating the number of days with a score of 3
or higher for each group and comparing these numbers using a
chi-squared test. The results of this analysis are shown in Table
2. The hamsters in the saline treated control group had a score of
3 or higher on 46.9% of the animal days evaluated. In the group
receiving SCV-07 at 1 ug/kg from day -1 to 16, a mucositis score of
3 or higher also was observed on 46.9% of the animals days
evaluated. The hamsters in the groups treated with SCV-07 at 10
ug/kg on days -1 to 16 had a score of 3 or higher on 37.5% of
animal days respectively, which was not statistically significantly
different from the controls (P=0.079). The group treated with
SCV-07 at 100 ug/kg on days -1 to 16 on days 1 to 16 had a score of
3 or higher on 35.4% of animal days, which was significantly
different from controls (P=0.029).
[0048] A further analysis of the mucositis scores was performed
using the Mann-Whitney rank sum analysis to compare the scores for
each group on each day. The results of this analysis are shown in
Table 4. In this analysis, 2 days of significant reduction in the
mucositis score are generally required before it is regarded as
meaningful reduction in mucositis. The group treated with SCV-07 at
1 ug/kg on days -1 to 16 did not show any statistically significant
improvement on any day of the study, relative to the saline
controls. The group treated with SCV-07 at 10 ug/kg on days -1 to
16 showed significant improvement relative to controls on day 22
(P=0.024). The group treated with SCV-07 at 100 ug/kg on days -1 to
16 showed significant improvement relative to controls on day 14
(P=0.025).
TABLE-US-00003 TABLE 3 Chi-square analysis of the total number of
days the animals in each group spent with a score of three or more.
This statistic is a measure of severity of ulceration, a clinically
important outcome. Days Days Total % Days Chi Sq Group >=3 <3
Days >=3 v control P Value Control 90 102 192 46.9 -- -- SCV-07
90 102 192 46.9 0.0105 0.919 1 ug/kg sc Days -1 to 16 SCV-07 72 120
192 37.5 3.0860 0.079 10 ug/kg sc Days -1 to 16 SCV-07 68 124 192
35.4 4.7420 0.029 100 ug/kg sc Days -1 to 16
TABLE-US-00004 TABLE 4 The significance of group differences
observed in daily mucositis scores was determined using the
Mann-Whitney rank sum test. This nonparametric statistic is
appropriate for the visual mucositis scoring scale. The p values
for each calculation are shown. Significant improvements are shown
underlined. Day Group Comparison 6 8 10 12 14 16 18 20 22 24 26 28
Control vs 0.555 0.775 0.069 0.531 0.234 0.145 0.557 0.373 0.230
0.558 0.554 0.733 SCV-07 1 ug/kg Days -1 to 16 Control vs 0.985
0.775 0.372 0.554 0.117 0.265 0.374 0.116 0.024 0.316 0.608 0.361
SCV-07 10 ug/kg Days -1 to 16 Control vs 0.555 0.985 0.776 0.261
0.025 0.061 0.112 0.100 0.461 0.192 0.484 0.531 SCV-07 100 ug/kg
Days -1 to 16
Conclusions
[0049] 1. There was no evidence of any toxicity from SCV-07 in this
study based on the observations of mortality and weight gain. 2.
Animals treated with SCV-07 at 10 ug/kg on days -1 to 16 showed a
statistically significant reduction in mucositis scores on day 22
(P=0.024) that was dose related. 3. Animals treated with SCV-07 at
100 ug/kg on days -1 to 16 showed a statistically significant
reduction in mucositis scores on day 14 (P=0.025), in addition to a
statistically significant reduction in the number of animal days
with a score of 3 or higher (P=0.029). 4. A favorable
dose-dependent effect of SCV-07 on the severity and course of
mucositis was seen. Increasing the dosing frequency or doubling the
single daily dose might enhance the effects noted.
APPENDICES
TABLE-US-00005 [0050] APPENDIX 1 Animal Weights DAY Group Animal -1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 77 80 78 83 85 86 91 92 96
96 98 97 102 102 101 102 1 2 87 90 92 97 99 101 106 110 113 119 118
119 124 125 125 127 1 3 89 92 91 96 99 102 107 108 113 115 111 118
126 126 122 123 1 4 81 82 82 86 87 89 94 96 96 99 102 101 107 107
106 106 1 5 83 86 86 90 89 91 94 101 99 101 106 102 108 108 108 109
1 6 82 85 87 92 94 96 99 104 106 106 112 108 110 110 111 110 1 7 82
93 93 98 102 104 109 113 115 117 120 119 124 127 126 125 1 8 88 80
79 83 84 86 92 92 97 98 100 100 103 105 104 105 2 9 78 96 98 100
101 103 109 111 114 116 118 117 122 123 122 123 2 10 94 86 86 89 91
94 97 99 99 102 104 104 109 110 110 110 2 11 93 80 79 84 88 90 91
95 96 101 101 100 104 105 104 107 2 12 79 83 83 86 86 89 94 98 100
103 104 102 105 107 109 111 2 13 82 86 88 91 93 95 100 105 103 105
107 106 111 114 112 116 2 14 84 86 88 91 93 96 100 101 104 105 107
106 111 114 111 112 2 15 79 80 81 84 84 86 91 94 94 97 99 98 105
110 108 110 2 16 79 81 78 83 84 87 89 93 94 98 100 97 102 105 104
106 3 17 81 83 84 87 89 92 96 98 100 102 104 103 111 112 111 111 3
18 87 90 91 94 98 100 105 107 108 111 112 113 117 120 118 115 3 19
85 87 87 90 92 93 97 98 101 103 105 104 109 109 109 103 3 20 76 76
76 79 82 84 89 90 92 96 96 96 101 101 100 102 3 21 90 93 93 96 98
100 103 106 108 109 111 110 113 117 113 110 3 22 91 95 94 98 100
103 108 109 112 114 116 115 121 124 122 122 3 23 86 91 91 95 98 100
105 101 109 113 114 115 121 121 121 121 3 24 91 95 96 100 104 106
113 116 115 120 122 124 129 130 130 129 4 25 84 87 87 91 93 96 100
103 105 109 111 110 117 119 117 117 4 26 87 91 98 94 95 98 101 104
105 109 112 111 119 117 115 118 4 27 97 101 101 108 110 111 119 123
124 127 132 133 137 138 134 139 4 28 88 95 93 98 101 103 101 115
118 118 124 121 130 131 132 139 4 29 86 90 86 92 97 100 104 106 108
111 114 115 120 123 121 125 4 30 84 88 89 92 95 99 106 109 111 115
120 118 129 131 127 132 4 31 95 100 99 106 109 111 118 119 122 126
127 128 136 137 133 138 4 32 85 87 89 94 94 97 105 106 108 110 114
113 118 121 118 120 DAY Group Animal 15 16 17 18 19 20 21 22 23 24
25 26 27 28 1 1 101 103 100 107 109 109 112 113 116 115 119 119 121
123 1 2 126 125 130 134 140 141 143 146 149 149 153 152 154 158 1 3
122 129 123 130 134 135 137 140 142 147 147 148 149 152 1 4 106 109
106 115 116 117 119 121 126 125 130 127 131 136 1 5 108 110 109 118
119 121 122 124 129 128 131 132 135 137 1 6 111 113 113 120 121 121
124 127 128 130 135 135 138 140 1 7 127 128 127 137 139 141 143 144
149 149 153 153 156 159 1 8 105 108 106 114 117 117 119 123 126 126
129 129 132 135 2 9 123 129 126 133 136 137 139 141 143 143 147 148
150 152 2 10 110 115 114 118 121 120 122 125 128 129 131 134 135
138 2 11 111 112 108 117 118 118 121 123 124 124 129 131 134 135 2
12 111 116 112 119 123 122 123 124 130 131 137 133 139 138 2 13 117
121 117 125 127 128 131 132 135 133 140 140 144 147 2 14 112 115
113 119 120 122 123 125 126 128 131 131 133 136 2 15 109 113 108
116 118 119 120 123 125 126 130 130 133 137 2 16 106 111 109 115
118 120 121 121 126 125 129 131 132 135 3 17 112 116 116 122 124
123 122 123 126 126 132 132 133 137 3 18 110 102 107 113 115 117
119 121 122 126 128 130 133 137 3 19 101 111 102 107 111 112 113
115 117 120 122 123 120 129 3 20 105 107 104 113 116 115 119 119
122 122 127 127 131 134 3 21 111 115 111 117 119 120 120 123 125
125 128 129 132 135 3 22 120 124 123 130 132 134 136 137 140 139
145 145 149 151 3 23 121 124 125 132 137 139 139 141 145 146 146
153 155 137 3 24 131 134 133 142 146 147 149 150 155 155 160 161
165 167 4 25 116 118 118 124 124 124 123 124 124 124 127 127 129
131 4 26 117 120 119 126 126 129 132 134 135 135 138 140 143 147 4
27 138 141 142 151 153 154 156 157 161 167 167 175 169 172 4 28 138
143 143 152 155 159 159 162 164 167 173 169 175 182 4 29 126 129
130 137 139 141 143 147 151 150 156 158 160 166 4 30 133 136 136
144 149 148 150 154 156 160 165 165 167 168 4 31 139 142 140 148
151 152 153 156 156 157 164 163 164 166 4 32 119 125 125 131 134
136 137 138 141 142 147 147 149 152
TABLE-US-00006 APPENDIX 2 Mucositis Scores Ani- DAYS Group mal 6 8
10 12 14 16 18 20 22 24 26 28 1 1 0 1 1 1 2 3 4 4 3 2 2 1 1 2 0 1 1
1 2 3 4 3 3 3 2 1 1 3 0 1 2 2 3 3 4 3 2 2 2 1 1 4 1 1 1 2 2 3 4 3 3
3 2 1 1 5 0 1 1 2 2 3 4 3 2 2 1 1 1 6 0 0 1 2 2 3 4 3 3 3 3 1 1 7 0
0 1 3 2 3 4 3 3 2 1 1 1 8 0 0 1 2 3 4 4 3 2 2 1 1 2 9 0 1 2 2 3 4 4
3 3 3 2 0 2 10 1 1 1 2 2 3 4 3 3 3 3 1 2 11 0 1 1 2 3 3 4 3 3 2 2 1
2 12 0 0 2 3 3 3 4 3 2 3 2 1 2 13 1 1 3 2 3 3 3 2 2 2 2 1 2 14 0 1
1 2 2 3 4 3 3 3 2 1 2 15 0 1 1 1 2 3 4 4 2 2 2 1 2 16 0 1 1 2 2 3 4
3 2 2 1 1 3 17 1 1 1 2 2 3 4 3 2 2 2 1 3 18 0 0 1 2 3 3 4 3 2 2 2 2
3 19 1 1 1 2 3 3 4 3 2 3 2 1 3 20 1 1 1 2 2 3 3 2 1 2 1 1 3 21 1 1
1 2 2 3 4 3 3 3 3 1 3 22 0 1 1 1 2 3 4 3 2 2 2 1 3 23 0 1 1 2 2 3 4
3 1 2 2 1 3 24 1 1 1 2 2 3 3 3 2 2 1 0 4 25 0 1 1 1 2 3 4 4 4 3 3 1
4 26 0 1 1 2 2 3 4 3 3 2 2 1 4 27 0 0 2 2 2 3 4 3 3 2 3 2 4 28 1 1
1 2 2 3 3 3 2 2 3 3 4 29 0 0 1 2 3 3 4 3 2 2 1 1 4 30 1 1 1 2 2 2 2
1 2 2 1 1 4 31 0 0 1 2 2 3 3 2 1 2 1 1 4 32 1 0 1 2 3 4 4 3 3 2 1
1
Example 2
A Second Study of SCV-07 in the Treatment of Oral Mucositis Induced
by Acute Radiation in Hamsters
[0051] The objective of this study was to conduct a preliminary
evaluation of the efficacy of SCV-07 in the treatment of oral
mucositis using an acute radiation induced hamster model of the
disease. SCV-07 was given at doses of 1 mg/kg or 100 .mu.g/kg once
or twice daily by sub-cutaneous injection for 22 days, starting one
day before radiation and continuing until day 20 after radiation.
No deaths were observed in any of the treatment groups and there
were statistically significant increases in growth rate, suggesting
that SCV-07 was well tolerated at these doses, and may actually
reduce the weight loss associated with mucositis. Control hamsters
had a mucositis score of 3 or higher on 28.1% of the animal days
evaluated in this study. Animals treated with SCV-07 at 100
.mu.g/kg once daily on days -1 to 20 showed a statistically
significant reduction in the number of animals days with a score of
3 or higher to 6.3% (P<0.001), and a statistically significant
reduction in individual daily scores on days 14 (P=0.011), 16
(P=0.002) and 18 (P=0.001). Animals treated with SCV-07 at 100
.mu.g/kg twice daily on days -1 to 20 showed a statistically
significant reduction in the number of animals days with a score of
3 or higher to 8.9% (P<0.001), and a statistically significant
reduction in individual daily scores on days 18 (P<0.001) and 20
(P=0.003). Animals treated with SCV-07 at 1 mg/kg twice daily on
days -1 to 20 showed a statistically significant reduction in the
number of animals days with a score of 3 or higher to 12.5%
(P<0.001), and a statistically significant reduction in
individual daily scores on days 16 (P=0.043) 18 (P=0.009) and 20
(P=0.007). Animals treated with SCV-07 at 1 mg/kg twice daily on
days -1 to 20 did not show any significant reduction in mucositis
scores. There was no statistically significant difference in the
number of animal days with a score of 3 or higher between the group
treated with SCV-07 at 100 .mu.g/kg once daily and the group
treated with SCV-07 at 1 mg/kg twice daily (P=0.054). When
individual daily scores were compared between these two groups, a
single day of significant difference was observed on day 14
(P=0.005). These observations suggest that treatment with SCV-07 at
100 .mu.g/kg once daily is very close to being significantly better
that treatment with SCV-07 at 1 mg/kg twice daily. This observation
suggests that 100 .mu.g/kg once daily is the dose closest to the
optimal dose of the doses tested in this study and in the prior
study SCI-01.
Introduction
1.1 Background
[0052] Oral ulcerative mucositis is a common, painful,
dose-limiting toxicity of drug and radiation therapy for cancer.
The disorder is characterized by breakdown of the oral mucosa that
results in the formation of ulcerative lesions. In granulocytopenic
patients, the ulcerations that accompany mucositis are frequent
portals of entry for indigenous oral bacteria often leading to
sepsis or bacteremia. Mucositis occurs to some degree in more than
one third of patients receiving anti-neoplastic drug therapy. The
frequency and severity are significantly greater among patients who
are treated with induction therapy for leukemia or with many of the
conditioning regimens for bone marrow transplant. Among these
individuals, moderate to severe mucositis is not unusual in more
than three-quarters of patients. Moderate to severe mucositis
occurs in virtually all patients who receive radiation therapy for
tumors of the head and neck and typically begins with cumulative
exposures of 15 Gy and then worsens as total doses of 60 Gy or more
are reached.
[0053] Clinically mucositis progresses through three stages:
1. Inflammation accompanied by painful mucosal erythema, which can
respond to local anesthetics. 2. Painful ulceration with
pseudomembrane formation and, in the case of myelosuppressive
treatment, potentially life-threatening sepsis, requiring
antimicrobial therapy. Pain is often of such intensity as to
require parenteral narcotic analgesia. 3. Spontaneous healing,
occurring about 2-3 weeks after cessation of anti-neoplastic
therapy.
[0054] Standard therapy for mucositis is predominantly palliative,
including application of topical analgesics such as lidocaine
and/or systemic administration of narcotics and antibiotics.
Currently, there is only one approved treatment for oral mucositis,
Kepivance (Palifermin), which is only approved for the treatment of
oral mucositis in patients undergoing conditioning regimens prior
to hematopoetic stem cell transplantation for the treatment of
hematologic malignancies.
[0055] The complexity of mucositis as a biological process has only
been recently appreciated. It has been suggested that the condition
represents a sequential interaction of oral mucosal cells and
tissues, reactive oxygen species, pro-inflammatory cytokines,
mediators of apoptosis and local factors such as saliva and the
oral micro biota. While epithelial degeneration and breakdown
ultimately result in mucosal ulceration, it appears that the early
changes associated with radiation-induced mucosal toxicity occur
within the endothelium, and connective tissue of the submucosa.
Electron microscopic evaluation of mucosa within 1 week of
radiation shows damage to both endothelium and connective tissue,
but not epithelium. Such injury is likely mediated by free radical
formation. It appears that the overall mechanism for mucositis
development is similar for both radiation and chemotherapy.
1.2 Acute Radiation Model
[0056] The acute radiation model in hamsters, developed by the
Principle Investigator, has proven to be an accurate, efficient and
cost-effective technique to provide a preliminary evaluation of
anti-mucositis compounds. The course of mucositis in this model is
well defined and results in peak scores approximately 14-16 Days
following radiation. The acute model has little systemic toxicity,
resulting in few hamster deaths, thus permitting the use of smaller
groups (N=7-8) for initial efficacy studies. It has also been used
to study specific mechanistic elements in the pathogenesis of
mucositis. Molecules that show efficacy in the acute radiation
model may be further evaluated in the more complex models of
fractionated radiation, chemotherapy, or concomitant therapy.
[0057] In this study, an acute radiation dose of 40 Gy on day 0 was
administered. Clinically significant mucositis was observed on days
12 through 28.
2. Study Objective and Summary
2.1 Study Objective
[0058] The objective of this study was to evaluate the effect of
SCV-07, administered by sub-cutaneous injection on different
schedules between the day before radiation and the twentieth day
after radiation, on the frequency, severity and duration of oral
mucositis induced by acute radiation. A previous study with SCV-07
(Study SCI-01, Example 1), had indicated some activity against
mucositis at a dose of 100 .mu.g/kg, given once daily from day -1
to day 16. In this study, SCV-07 was dosed at 100 .mu.g/kg and 1
mg/kg once or twice daily from day -1 to day 20, to see if the
partial effect seen in the previous study could be extended.
2.2 Study Summary
[0059] Forty (40) Syrian Golden Hamsters were given an acute
radiation dose of 40 Gy directed to their left buccal cheek pouch
on day 0. Test material SCV-07 was given by sub-cutaneous injection
at 100 .mu.g/kg or 1 mg/kg once or twice daily. Dosing began one
day before radiation (day -1) and continued until day 20. Mucositis
was evaluated clinically starting on day 6, and continuing on
alternate days until day 28.
3. Evaluation
3.1 Mucositis Evaluation
[0060] The grade of mucositis was scored, beginning day 6, and for
every second day thereafter, through and including day 28. The
effect on mucositis of each drug treatment compared to placebo was
assessed according to the following parameters:
3.1.1 The Difference in the Number of Days Hamsters in Each Group
have Ulcerative (score .gtoreq.3) mucositis.
[0061] On each evaluation day, the number of animals with a blinded
mucositis score of .gtoreq.3 in each drug treatment group was
compared to the control group. Differences were compared on a
cumulative basis and statistical significance was determined by
chi-square analysis. Efficacy, in this analysis, is defined by a
significant reduction in the number of days that a group of animals
had ulcerations (scores .gtoreq.3) when compared to the control
group. This same test was also used to evaluate differences between
different drug treatment groups.
3.1.2 Rank Sum Differences in Daily Mucositis Scores.
[0062] For each evaluation day the scores of the control group were
compared to those of the treated groups using non-parametric rank
sum analysis. Treatment success was considered as a statistically
significant lowering of scores in the treated group on 2 or more
days from day 6 to day 28.
3.2 Weights and Survival
[0063] All animals were weighed daily and their survival recorded,
in order to assess possible differences in animal weight among
treatment groups as an indication for mucositis severity and/or
possible toxicity resulting from the treatments.
4. Study Design
[0064] Forty Syrian Golden Hamsters were divided into five (5)
groups of eight (8) animals each. All animals received a single
dose of acute radiation of 40 Gy directed to their left buccal
cheek pouch on day 0. This was accomplished by anesthetizing the
animals and everting the left buccal pouch, while protecting the
rest of the animals with a lead shield. Test materials were given
by sub-cutaneous injection once daily as detailed in Table 5.
Mucositis was evaluated clinically starting on Day 6, and
continuing on alternate days until day 28.
TABLE-US-00007 TABLE 5 SCI-02. Study Design Group Number of
Treatment Volume Number Animals Treatment Schedule* (mL) 1 8 males
Vehicle (PBS), sc, qd Day -1 to 20 Adjust per body weight 2 8 males
SCV-07, sc, qd Day -1 to 20 Adjust per 100 .mu.g/kg, body weight 3
8 males SCV-07, sc, bid Day -1 to 20 Adjust per 100 .mu.g/kg, body
weight 4 8 males SCV-07, sc, qd Day -1 to 20 Adjust per 1.0 mg/kg
body weight 5 8 males SCV-07, sc, bid Day -1 to 20 Adjust per 1.0
mg/kg body weight *The dose on day 0 was performed 30 minutes prior
to radiation
5. Material and Methods
5.1 Location of Study Performance
[0065] The study was performed at Biomodels AAALAC accredited
facility in Watertown Mass. Approval for this study was obtained
from Biomodels IACUC.
5.2 Animals
[0066] Male LVG Syrian Golden Hamsters (Charles River
Laboratories), aged 5 to 6 weeks, with average body weight of 81.9
g at study commencement, were used. Animals were individually
numbered using an ear punch and housed in small groups of 8 animals
per cage. Animals were acclimatized prior to study commencement.
During this period of 5 days, the animals were observed daily in
order to reject animals that presented in poor condition.
5.3 Housing
[0067] 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. Bedding was
changed a minimum of once per week. Cages, tops, bottles, etc. were
washed with a commercial detergent and allowed to air dry. 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 marked all cages. The temperature and relative
humidity was recorded during the study, and the records
retained.
5.4 Diet
[0068] Animals were fed with a Purina Labdiet.RTM. 5061 rodent diet
and water was provided ad libitum.
5.5 Animal Randomization and Allocations.
[0069] Animals were randomly and prospectively divided into five
(5) treatment groups prior to irradiation. Each animal was
identified by an ear punch corresponding to an individual number.
For more consistent identification, ear punch numbering was used
rather than tagging, since tags may become dislodged during the
course of the study. A cage card was used to identify each cage or
label marked with the study number (SCI-02), treatment group number
and animal numbers.
5.6 Sub-Cutaneous Dosing and Drug Application
[0070] The test compound, SCV-07 was provided as a powder and
dissolved in sterile PBS immediately prior to administration. Three
dilutions were prepared: 100 .mu.g/mL, 10 .mu.g/mL and 1 .mu.g/mL.
Drug was given in a volume of 0.1 mL per 100 g body weight, with
the appropriate dilution of SCV-07 for each group, using a
tuberculin syringe with a 27G needle. Injections were given
subcutaneously to the back or abdomen.
5.7 Mucositis Induction
[0071] Mucositis was induced using a standardized acute radiation
protocol. A single dose of radiation (40 Gy/dose) was administered
to all animals on day 0. Radiation was generated with a 160
kilovolt potential (15-ma) source at a focal distance of 30 cm,
hardened with a 0.35 mm Cu filtration system. Irradiation targeted
the left buccal pouch mucosa at a rate of 3.2 Gy/minute. Prior to
irradiation, animals were anesthetized with an intraperitoneal
injection of Ketamine (160 mg/kg) and Xylazine (8 mg/kg). The left
buccal pouch was everted, fixed and isolated using a lead shield to
protect all other parts of the hamster's body.
5.8 Mucositis Scoring
[0072] The mucositis score, weight change and survival were
measured throughout the study as described above. For the
evaluation of mucositis, the animals were anesthetized with an
inhalation anesthetic, and the left pouch everted. Mucositis was
scored visually by comparison to a validated photographic scale,
ranging from 0 for normal, to 5 for severe ulceration (clinical
scoring). In descriptive terms, this scale is defined as
follows:
TABLE-US-00008 TABLE 6 SCI-02: Mucositis Scoring. Score:
Description: 0 Pouch completely healthy. No erythema or
vasodilation. 1 Light to severe erythema and vasodilation. No
erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of
superficial aspects of mucosa leaving denuded areas. Decreased
stippling of mucosa. 3 Formation of off-white ulcers in one or more
places. Ulcers may have a yellow/gray due to pseudomembrane.
Cumulative size of ulcers should equal about 1/4 of the pouch.
Severe erythema and vasodilation. 4 Cumulative seize of ulcers
should equal about 1/2 of the pouch. Loss of pliability. Severe
erythema and vasodilation. 5 Virtually all of pouch is ulcerated.
Loss of pliability (pouch can only partially be extracted from
mouth).
[0073] A score of 1-2 is considered to represent a mild stage of
the disease, whereas a score of 3-5 is considered to indicate
moderate to severe mucositis. Following visual scoring, a
photograph was taken of each animal's mucosa using a standardized
technique. At the conclusion of the experiment, all films were
developed and the photographs randomly numbered. At least two
independent trained observers graded the photographs in blinded
fashion using the above-described scale (blinded scoring).
6. Results and discussion
6.1 Survival
[0074] No deaths were observed during the course of this study.
6.2 Weight Change
[0075] The mean daily percent weight change data was evaluated. The
saline treated control hamsters gained an average of 44.1% of their
starting weight during the study. Hamsters in the group receiving
SCV-07 100 .mu.g/kg once daily on days -1 to 20 gained an average
of 49.9% of their starting weight during the study. Hamsters in the
group receiving SCV-07 at 100 .mu.g/kg twice daily on days -1 to 20
gained an average of 61.3% of their starting weight during the
study. Hamsters in the group receiving SCV-07 at 1 mg/kg once daily
on days -1 to 20 gained an average of 63.4% of their starting
weight during the study. Hamsters in the group receiving SCV-07 at
1 mg/kg twice daily on days -1 to 20 gained an average of 69.1% of
their starting weight during the study. The significance of these
differences was evaluated by calculating the area-under-the-curve
(AUC) for the weight gain of each animal, and then comparing the
different treatment groups using a One-Way ANOVA test. The results
of this analysis indicated that there were significant differences
between the SCV-07 treated groups and the control group (P=0.012).
The groups treated with SCV-07 100 .mu.g/kg twice daily, 1 mg/kg
once and twice daily all had significantly greater weight gain than
the saline controls (P=0.014, P=0.009 and P=0.004
respectively).
6.3 Mucositis (Tables 7 & 8)
[0076] Mean daily mucositis scores for each group are were
determined. In the saline treated control group, peak levels of
mucositis were seen on day 18, when the mean score reached 3.0. The
group receiving SCV-07 at 100 .mu.g/kg once daily from day -1 to 20
had a peak mean mucositis score of 2.2 on day 16. The group
receiving SCV-07 at 100 .mu.g/kg twice daily from day -1 to 20 had
a peak mean mucositis score of 2.5 which occurred on days 14 and
16. The group receiving SCV-07 at 1 mg/kg once daily from day -1 to
20 had a peak mean mucositis score of 2.9 on day 14. The group
receiving SCV-07 at 1 mg/kg twice daily from day -1 to 20 had a
peak mean mucositis score of 2.6 on day 14. The significance of the
differences observed between the different treatment groups was
evaluated by calculating the number of days with a score of 3 or
higher for each group and comparing these numbers using a
chi-squared (.quadrature..sup.2) test. The results of this analysis
are shown in Table 7. The hamsters in the saline treated control
group had a score of 3 or higher on 28.1% of the animal days
evaluated. In the group receiving SCV-07 at 100 .mu.g/kg once daily
from day -1 to 20, a mucositis score of 3 or higher was observed on
6.3% of the animals days evaluated, which was statistically
significantly different that the controls (P<0.001). The
hamsters in the groups treated with SCV-07 at 100 .mu.g/kg twice
daily on days -1 to 20 had a score of 3 or higher on 8.9% of animal
days respectively, which was statistically significantly different
from the controls (P<0.001). The group treated with SCV-07 at 1
mg/kg once daily on days -1 to 20 had a score of 3 or higher on
28.1% of animal days, which was not significantly different from
controls (P=1.000). The group treated with SCV-07 at 1 mg/kg twice
daily on days -1 to 20 had a score of 3 or higher on 12.5% of
animal days, which was significantly different from controls
(P<0.001). There was no statistically significant difference in
the number of animal days with a score of 3 or higher between the
group treated with SCV-07 at 100 .mu.g/kg once daily and the group
treated with SCV-07 at 1 mg/kg twice daily (P=0.054).
[0077] A further analysis of the mucositis scores was performed
using the Mann-Whitney rank sum analysis to compare the scores for
each group on each day. The results of this analysis are shown in
Table 8. In this analysis, 2 days of significant reduction in the
mucositis score are generally required before it is regarded as
meaningful reduction in mucositis. The group treated with SCV-07 at
100 .mu.g/kg once daily on days -1 to 20 showed statistically
significant improvement on days 14 (P=0.011), 16 (P=0.002) and 18
(P=0.001) of the study relative to the saline controls. The group
treated with SCV-07 at 100 .mu.g/kg twice daily on days -1 to 20
showed significant improvement relative to controls on days 18
(P<0.001) and 20 (P=0.003). The group treated with SCV-07 at 1
mg/kg once daily on days -1 to 20 showed no significant improvement
in mucositis on any day of the study relative to controls. The
group treated with SCV-07 at 1 mg/kg twice daily on days -1 to 20
showed significant improvement relative to controls on days 16
(P=0.043) 18 (P=0.009) and 20 (P=0.007). A comparison between the
group treated with SCV-07 at 100 .mu.g/kg once daily and the group
treated with SCV-07 at 1 mg/kg twice daily showed a single day of
statistically significant difference on day 14 (P=0.005).
TABLE-US-00009 TABLE 7 SCI-02. Chi-square analysis of the total
number of days the animals in each group spent with a score of
three or more. This statistic is a measure of severity of
ulceration, a clinically important outcome. Days Days Total % Days
Chi Sq Group >=3 <3 Days >=3 v control P Value Vehicle
(PBS), qd, 54 138 192 28.1 -- -- sc SCV-07 12 180 192 6.3 27.828
<0.001 100 ug/kg, qd, sc SCV-07 17 175 192 8.9 21.4010 <0.001
100 ug/kg, bid, sc SCV-07 54 138 192 28.1 0.0000 1.000 1 mg/kg, qd,
sc SCV-07 24 168 192 12.5 12.7360 <0.001 1 mg/kg, bid, sc
TABLE-US-00010 TABLE 8 SCI-02. The significance of group
differences observed in daily mucositis scores was determined using
the Mann-Whitney rank sum test. This nonparametric statistic is
appropriate for the visual mucositis scoring scale. The p values
for each calculation are shown. Significant improvements are shown
underlined. Day Group Comparison 6 8 10 12 14 16 18 20 22 24 26 28
Control vs 0.558 0.985 0.985 0.425 0.011 0.002 0.001 0.092 0.413
0.436 0.555 0.374 SCV-7 100 ug/kg, qd, sc Days -1 to 20 Control vs
0.558 0.554 0.985 0.581 0.664 0.204 <0.001 0.003 0.279 0.805
0.775 0.558 SCV-7 100 ug/kg, bid, sc Days -1 to 20 Control vs 0.233
0.985 0.985 0.775 0.218 0.662 0.116 0.405 0.404 0.298 0.774 0.895
SCV-7 1 mg/kg, qd, sc Days -1 to 20 Control vs 0.072 0.985 0.985
0.805 0.925 0.043 0.009 0.007 0.581 0.985 0.774 0.777 SCV-7 1
mg/kg, bid, sc Days -1 to 20 SCV-7 100 ug/kg, qd, sc vs 0.233 0.985
0.985 0.279 0.005 0.232 0.335 0.371 0.775 0.370 0.370 0.557 SCV-7 1
mg/kg, bid, sc Days -1 to 20
7. Conclusions
[0078] 1. There was no evidence of any toxicity from SCV-07 in this
study based on the observations of mortality and weight gain. 2.
The groups treated with SCV-07 at 100 .mu.g/ml twice daily or
SCV-07 1 mg/ml once or twice daily showed significant increases in
weight gain relative to the saline controls (P=0.014, P=0.009 and
P=0.004 respectively). 3. Animals treated with SCV-07 at 100
.mu.g/kg once daily on days -1 to 20 showed a statistically
significant reduction in the number of animals days with a score of
3 or higher (P<0.001), and a statistically significant reduction
in individual daily scores on days 14 (P=0.011), 16 (P=0.002) and
18 (P=0.001). 4. Animals treated with SCV-07 at 100 .mu.g/kg twice
daily on days -1 to 20 showed a statistically significant reduction
in the number of animals days with a score of 3 or higher
(P<0.001), and a statistically significant reduction in
individual daily scores on days 18 (P<0.001) and 20 (P=0.003).
5. Animals treated with SCV-07 at 1 mg/kg twice daily on days -1 to
20 showed a statistically significant reduction in the number of
animals days with a score of 3 or higher (P<0.001), and a
statistically significant reduction in individual daily scores on
days 16 (P=0.043) 18 (P=0.009) and 20 (P=0.007). 6. Animals treated
with SCV-07 at 1 mg/kg twice daily on days -1 to 20 did not show
any significant reduction in mucositis scores. 7. There was no
statistically significant difference in the number of animal days
with a score of 3 or higher between the group treated with SCV-07
at 100 .mu.g/kg once daily and the group treated with SCV-07 at 1
mg/kg twice daily (P=0.054). When individual daily scores were
compared between these two groups, a single day of significant
difference was observed on day 14 (P=0.005).
[0079] These observations suggest that treatment with SCV-07 at 100
.mu.g/kg once daily is very close to being significantly better
that treatment with SCV-07 at 1 mg/kg twice daily.
9. Appendices
TABLE-US-00011 [0080] APPENDIX 3 Animal Weights Group Animal 0 1 2
3 4 5 6 7 8 9 10 11 12 13 14 15 1 1 84.7 84.6 87 89 92 94 96 98 98
102 104 105 107 104 106 105 1 2 79.1 79 79 83 88 88 92 91 93 101
101 102 104 105 108 108 1 3 84.1 84.3 86 88 93 93 95 97 97 102 104
105 115 105 105 103 1 4 81.3 79.1 81 82 84 84 86 89 90 92 90 96 99
97 99 100 1 5 84.6 86.9 86 89 93 95 98 100 99 106 108 109 110 109
108 108 1 6 90.9 92.3 94 96 99 103 107 108 107 113 113 113 105 113
117 116 1 7 80.3 78.5 82 83 86 89 91 92 95 97 100 100 103 99 101
101 1 8 85.3 85.2 89 90 95 98 99 100 105 108 108 108 109 109 111
110 2 9 87.6 86.5 92 96 98 102 105 107 108 114 117 109 120 118 124
123 2 10 87.2 80.5 86 94 97 99 101 103 105 111 110 103 114 110 116
116 2 11 81.6 81.7 86 86 87 88 90 90 94 95 97 90 98 95 98 99 2 12
71.9 72.8 75 77 79 79 85 84 85 88 91 88 97 97 101 98 2 13 86.2 85.7
91 93 94 95 98 97 99 101 105 98 109 104 110 110 2 14 81.9 82.6 86
87 90 90 92 94 94 101 100 95 104 102 106 102 2 15 82.7 83.4 88 91
93 95 97 99 100 105 106 101 109 106 110 111 2 16 80.4 81.2 90 88 89
92 93 94 98 98 103 99 107 106 111 113 3 17 77.1 77.1 80 83 79 84 91
91 85 98 100 99 101 97 103 104 3 18 75 75.8 82 81 81 82 85 85 81 90
92 91 92 88 94 94 3 19 86.6 86.1 93 94 92 98 102 105 99 113 115 117
120 116 121 127 3 20 80.2 79.1 86 89 84 86 93 95 89 100 103 102 104
101 105 106 3 21 81.7 81 87 90 85 89 92 94 87 98 100 100 104 99 105
104 3 22 82 80.3 86 89 85 89 92 95 88 99 102 102 104 100 106 105 3
23 80.8 80.8 87 90 89 93 101 100 93 107 107 108 115 108 113 114 3
24 83 80.7 88 94 85 94 102 102 95 110 113 113 118 117 120 121 4 25
85.2 84.3 86 89 93 92 98 98 100 106 105 107 109 108 110 107 4 26
88.7 89.4 94 98 100 101 104 107 108 113 114 116 119 118 121 120 4
27 83.4 84.9 86 90 92 93 97 97 98 102 103 105 105 105 108 105 4 28
72.4 75.7 77 79 81 81 85 85 86 90 92 93 94 94 93 91 4 29 72.3 75.8
77 78 83 82 87 91 91 99 99 102 103 104 105 106 4 30 90.6 94 96 99
101 104 109 111 109 115 116 115 103 118 120 120 4 31 75.9 77.5 79
82 84 85 89 91 91 98 99 100 117 103 105 105 4 32 84.8 86.4 91 95 99
98 103 104 102 109 111 111 116 116 118 116 5 33 90.1 87.6 89 93 94
98 106 106 107 112 113 113 117 118 119 118 5 34 76.5 77.3 78 79 81
81 83 86 87 90 91 90 92 93 117 99 5 35 87.5 89.6 91 95 98 98 105
106 109 114 116 118 122 119 95 120 5 36 81 82.8 84 89 96 93 101 100
101 106 107 107 110 113 122 114 5 37 74.7 74.2 83 79 81 85 87 88 91
95 97 98 99 99 100 99 5 38 79.8 81.8 81 87 90 90 94 96 98 104 104
105 109 108 112 112 5 39 77.3 76.6 75 85 87 88 90 92 94 97 99 99
102 103 106 107 5 40 80.1 78.8 80 83 86 86 95 96 97 99 102 100 102
102 104 104 Group Animal 16 17 18 19 20 21 22 23 24 25 26 27 28 1 1
109 104 102 109 110 113 113 117 118 119 118 118 116 1 2 112 106 108
116 115 122 122 127 129 129 128 132 126 1 3 107 101 99 107 108 111
111 114 116 119 117 118 114 1 4 105 99 97 105 104 108 125 112 112
114 114 115 111 1 5 112 106 107 113 113 118 121 121 123 125 124 123
123 1 6 120 112 111 120 119 127 127 132 134 134 133 134 132 1 7 105
97 95 105 107 111 113 118 117 121 118 121 120 1 8 1131 107 106 111
118 117 117 121 123 125 123 129 123 2 9 130 122 123 130 124 124 135
142 135 142 137 136 142 2 10 121 117 110 124 117 117 134 130 124
131 127 125 131 2 11 104 98 107 105 98 98 100 108 102 108 116 101
107 2 12 108 101 100 108 102 102 112 120 112 120 104 114 120 2 13
115 109 108 115 108 107 115 122 116 120 118 114 121 2 14 105 100 98
109 103 103 110 118 110 117 115 110 117 2 15 118 111 95 119 111 110
120 128 121 119 118 113 118 2 16 118 117 114 120 114 114 121 128
126 128 129 124 131 3 17 107 108 111 111 114 117 110 122 115 126
122 129 127 3 18 98 100 101 102 105 108 105 110 112 114 111 117 117
3 19 134 133 138 136 140 141 136 145 147 150 145 153 151 3 20 113
110 115 112 114 116 112 117 119 121 119 123 120 3 21 109 106 109
109 110 112 105 114 120 116 114 119 118 3 22 110 112 114 114 118
118 112 122 124 124 122 129 126 3 23 119 119 123 123 126 129 120
134 136 137 135 141 139 3 24 126 128 130 131 133 137 127 140 142
143 142 147 146 4 25 113 114 116 116 120 119 113 123 126 126 126
129 133 4 26 126 127 125 130 133 136 136 139 142 142 144 145 147 4
27 111 114 115 115 118 122 114 126 127 129 130 132 135 4 28 93 94
96 95 98 99 99 104 103 105 105 107 111 4 29 110 114 111 115 119 121
112 125 127 129 130 130 134 4 30 125 126 120 129 131 133 124 134
135 138 138 138 139 4 31 112 112 114 115 117 118 112 120 123 127
127 128 131 4 32 122 123 124 122 124 128 119 130 131 133 131 132
135 5 33 125 125 128 131 130 136 134 137 135 142 145 148 150 5 34
103 104 106 106 107 109 113 115 113 120 117 121 123 5 35 127 127
129 130 130 133 133 137 135 141 141 144 147 5 36 120 122 125 124
127 132 131 136 135 140 141 144 147 5 37 105 104 106 107 109 111
110 115 112 118 120 124 126 5 38 115 118 120 121 121 124 124 129
129 132 133 137 139 5 39 112 113 115 115 117 120 120 125 127 131
132 133 137 5 40 108 107 109 111 111 114 113 117 116 121 123 124
125
TABLE-US-00012 APPENDIX 4 Mucositis Scores Ani- DAY Group mal 6 8
10 12 14 16 18 20 22 24 26 28 1 1 0 1 2 2 2 3 4 3 2 2 2 2 1 1 0 1 1
2 2 3 4 3 2 2 2 1 1 2 0 1 2 3 3 3 3 2 2 1 1 1 1 2 0 1 1 3 3 3 3 2 2
2 2 1 1 3 0 1 2 2 3 3 3 3 2 2 2 2 1 3 0 1 1 2 3 3 3 3 2 2 2 1 1 4 0
1 2 2 1 1 2 1 1 1 2 2 1 4 0 1 1 1 2 1 2 2 2 2 2 1 1 5 0 1 2 2 3 3 3
3 2 2 2 2 1 5 0 1 1 2 3 3 3 3 2 2 2 1 1 6 1 1 2 2 2 3 3 3 2 2 2 2 1
6 1 1 1 2 2 3 3 3 2 2 2 1 1 7 1 1 2 2 3 3 3 2 2 2 2 2 1 7 1 1 1 2 3
3 3 2 2 2 2 1 1 8 1 1 2 2 3 3 3 3 3 3 2 2 1 8 1 1 1 2 3 3 3 3 3 3 2
2 2 9 1 1 2 2 2 2 2 2 1 1 1 1 2 9 1 1 1 2 2 2 2 2 2 2 2 1 2 10 1 1
2 3 2 2 2 2 2 2 2 2 2 10 1 1 1 2 2 2 2 2 2 2 2 1 2 11 1 1 2 2 2 2 3
3 2 2 1 2 2 11 1 1 1 2 2 2 3 3 2 2 2 1 2 12 0 1 2 2 2 2 1 2 2 1 2 1
2 12 0 1 1 2 2 2 2 2 2 2 2 1 2 13 0 1 2 2 2 2 1 1 1 1 2 1 2 13 0 1
1 1 1 2 2 2 2 2 2 1 2 14 0 1 2 2 3 3 3 3 2 2 2 2 2 14 0 1 1 1 2 3 3
3 2 2 2 1 2 15 1 1 2 2 2 2 2 2 2 2 2 2 2 15 1 1 1 2 2 2 2 2 2 2 2 1
2 16 0 1 2 2 2 2 2 2 2 2 1 2 2 16 0 1 1 1 2 2 2 2 2 2 2 1 3 17 0 1
2 2 2 3 2 2 1 2 2 1 3 17 0 1 1 2 2 3 2 2 2 2 2 1 3 18 1 1 2 2 3 3 3
2 2 2 2 2 3 18 1 1 1 2 3 3 2 2 2 2 2 1 3 19 1 1 2 1 2 1 1 1 1 1 1 1
3 19 1 1 1 2 2 2 2 2 2 2 2 1 3 20 1 1 2 2 3 3 2 2 2 2 2 2 3 20 1 1
1 2 3 3 2 2 2 2 2 1 3 21 1 1 2 2 2 3 2 1 1 2 2 2 3 21 1 1 1 2 2 2 2
2 2 2 2 1 3 22 0 1 2 2 2 2 2 2 2 2 1 2 3 22 0 1 1 2 2 2 2 2 2 2 2 1
3 23 0 0 2 2 3 2 2 2 2 2 2 2 3 23 0 0 1 2 3 2 2 2 2 2 2 1 3 24 0 1
2 2 3 3 2 2 2 2 2 2 3 24 0 1 1 2 3 3 2 2 2 2 2 1 4 25 1 1 2 2 3 3 3
3 3 3 3 3 4 25 1 1 1 2 3 3 3 3 3 3 2 1 4 26 0 1 2 2 3 3 3 3 3 3 2 2
4 26 0 1 1 2 3 3 3 3 3 3 2 1 4 27 1 1 2 2 3 3 3 3 3 3 2 2 4 27 1 1
1 2 3 3 3 2 2 2 2 1 4 28 0 1 2 2 3 3 3 2 2 2 2 2 4 28 0 1 1 2 3 3 3
3 2 2 2 1 4 29 1 1 2 2 3 3 3 3 2 2 2 2 4 29 1 1 1 2 3 3 3 2 2 2 2 1
4 30 0 1 2 2 3 2 2 2 2 2 2 2 4 30 0 1 1 2 3 2 2 2 2 2 2 1 4 31 1 1
2 2 3 3 2 2 2 2 2 2 4 31 1 1 1 2 3 3 2 2 2 2 2 1 4 32 1 1 2 2 2 2 2
1 1 1 1 2 4 32 1 1 1 2 2 2 2 2 2 2 2 1 5 33 1 1 2 2 2 2 2 2 2 2 2 2
5 33 1 1 1 2 2 2 2 2 2 2 2 1 5 34 1 1 2 2 3 2 2 2 2 2 2 2 5 34 1 1
1 2 3 2 2 2 2 2 2 1 5 35 1 1 2 2 3 3 3 2 2 2 2 2 5 35 1 1 1 2 3 3 3
2 2 2 2 1 5 36 0 1 2 2 2 2 2 2 2 2 2 1 5 36 0 1 1 2 2 2 2 2 2 2 2 1
5 37 0 1 2 2 3 2 2 2 2 2 2 2 5 37 0 1 1 2 3 2 2 2 1 2 2 1 5 38 1 1
2 2 3 2 2 2 2 2 2 2 5 38 1 1 1 2 3 2 2 2 2 2 2 1 5 39 1 1 2 2 2 3 3
2 2 2 2 2 5 39 1 1 1 2 2 3 3 2 2 2 2 1 5 40 1 1 2 3 3 3 3 2 2 2 2 2
5 40 1 1 1 3 3 3 3 2 2 2 2 1
Example 3
A Study of the Efficacy of Effect of SCV-07 in the Treatment of
Oral Mucositis Induced by Fractionated Radiation in Hamsters.
1. Introduction
1.1 Background
[0081] KGF-1 and other FGF family members have been shown to induce
epithelial thickening of the oral and esophageal mucosal surfaces
in BDF-1. SCV-07 and the derived SCV-07 peptide are believed to
have mechanisms that that may overlap with KGF-1, and have been
shown to be protective in other models of mucosal injury.
[0082] Oral ulcerative mucositis is a common, painful,
dose-limiting toxicity of drug and radiation therapy for cancer.
The disorder is characterized by breakdown of the oral mucosa that
results in the formation of ulcerative lesions. In granulocytopenic
patients, the ulcerations that accompany mucositis are frequent
portals of entry for indigenous oral bacteria often leading to
sepsis or bacteremia. Mucositis occurs to some degree in more than
one third of patients receiving anti-neoplastic drug therapy. The
frequency and severity are significantly greater among patients who
are treated with induction therapy for leukemia or with many of the
conditioning regimens for bone marrow transplant. Among these
individuals, moderate to severe mucositis is not unusual in more
than three-quarters of patients. Moderate to severe mucositis
occurs in virtually all patients who receive radiation therapy for
tumors of the head and neck and typically begins with cumulative
exposures of 15 Gy and then worsens as total doses of 60 Gy or more
are reached.
[0083] Clinically mucositis progresses through three stages:
1. Painful erythema which can generally be managed by topical
anesthetics or non-narcotic analgesics . . . . 2. Painful
ulceration often with pseudomembrane formation. In the case of
concomitant myelosuppression, bacteremias or sepsis of oral origin
are not uncommon. Pain is often of such intensity as to require
narcotic analgesia, frequently parenterally. 3. Spontaneous
healing, occurring about 2-3 weeks after cessation of
anti-neoplastic therapy.
[0084] Currently, the only approved biologic or drug for mucositis
prevention and/or treatment is Kepivance (palifermin). Kepivance
use is limited to mucositis in patients receiving stem cell
transplant for hematologic malignancies. Consequently, standard
therapy for mucositis consists of palliative rinses, such as
saline, bicarbonate solutions, mouthwashes, topical analgesics such
as lidocaine and/or systemic administration of narcotics.
[0085] The complexity of mucositis as a biological process has only
been recently appreciated. It has been suggested that the condition
represents a sequential interaction of oral mucosal cells and
tissues reactive oxygen species, pro-inflammatory cytokines,
mediators of apoptosis, a range of signaling pathways, and local
factors such as saliva and the oral micro biota. While epithelial
degeneration and breakdown ultimately result in mucosal ulceration,
it appears that the early changes associated with radiation-induced
mucosal toxicity occur within the endothelium, and connective
tissue of the submucosa. It appears that the overall mechanism for
mucositis development is similar for both radiation and
chemotherapy.
1.2 Fractionated Radiation Model
[0086] The fractionated radiation model in hamsters, developed by
the Principal Investigator, has proven to be an accurate, efficient
and cost-effective technique to provide a preliminary evaluation of
anti-mucositis compounds. In this model, hamsters receive 8 doses
of 7.5 Gy to the left cheek pouch on days 0, 1, 2, 3, 7, 8, 9 and
10, rather than the single dose of 40 Gy on day 0 that is used in
acute radiation studies. The rationale for using this scheduling
for the radiation is that it more closely resembles the clinical
courses of radiotherapy given to cancer patients. The course of
mucositis in this model is well defined and results in peak
mucositis scores approximately 14-16 days following radiation.
Mortality (generally from the side effects of anesthesia) in the
fractionated radiation model is slightly higher than that seen in
the acute radiation model and group sizes are increased (to 10 per
group) to allow for this.
2. Study Objective and Summary
2.1 Study Objective
[0087] The objective of this study was to evaluate the effect of
SCV-07, administered by sub-cutaneous injection, on the frequency,
severity and duration of oral mucositis induced by a fractionated
radiation protocol.
2.2 Study Summary
[0088] Forty (40) male Syrian Golden Hamsters were given eight
doses of radiation of 7.5 Gy each directed to their left buccal
cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9. This was
accomplished by anesthetizing the animals and everting the left
buccal pouch, while protecting the rest of the animal's bodies with
a lead shield. Test materials were given by sub-cutaneous injection
once daily as detailed in Table 9. Mucositis was evaluated
clinically starting on day 7, and continuing on alternate days
until day 35. Test articles were given as outlined Table 9, Groups
1 and 2 were dosed from Day -1 to Day 29, Group 3 was dosed on the
days of radiation only, Group 4 was dosed on the days between Day
-15 and Day 29 on which no radiation is given (Day -1, Day 4, Day 5
and Day 10 to Day 29).
3. Evaluation
3.1 Mucositis Evaluation
[0089] The grade of mucositis was scored, beginning day 6, and for
every second day thereafter, through and including day 34. The
effect on mucositis of each drug treatment compared to placebo was
assessed according to the following parameters:
3.1.1 The Difference in the Number of Days Hamsters in Each Group
have Ulcerative (Score .gtoreq.3) Mucositis.
[0090] On each evaluation day, the number of animals with a blinded
mucositis score of .gtoreq.3 in each drug treatment group was
compared to the control group. Differences were compared on a
cumulative basis and statistical significance was determined by
chi-square analysis. Efficacy, in this analysis, is defined by a
significant reduction in the number of days that a group of animals
had ulcerations (scores .gtoreq.3) when compared to the control
group.
3.1.2 Rank Sum Differences in Daily Mucositis Scores.
[0091] For each evaluation day the scores of the control group were
compared to those of the treated groups using non-parametric rank
sum analysis. Treatment success was considered as a statistically
significant lowering of scores in the treated group on 2 or more
days from day 6 to day 28.
3.2 Weights and Survival
[0092] All animals were weighed daily and their survival recorded,
in order to assess possible differences in animal weight among
treatment groups as an indication for mucositis severity and/or
possible toxicity resulting from the treatments.
4. Study Design
[0093] Forty (40) male Syrian Golden Hamsters were given eight
doses of radiation of 7.5 Gy each directed to their left buccal
cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9. This was
accomplished by anesthetizing the animals and everting the left
buccal pouch, while protecting the rest of the animal's bodies with
a lead shield. Test materials were given by sub-cutaneous injection
once daily at 8:00 am as detailed in Table 9. Mucositis was
evaluated clinically starting on Day 6, and continuing on alternate
days until day 34. Test articles were given as outlined Table 9,
Groups 1 and 2 was dosed from Day -1 to Day 29, Group 3 was dosed
on the days of radiation only (days 0-3 and days 6-10), Group 4 was
dosed on the days between Day -1 and Day 29 on which no radiation
is given (Day -1, Day 4 to Day 5 and Day 10 to Day 29).
TABLE-US-00013 TABLE 9 SCI-03. Study Design Number Number Group of
Treatment Treatment of Number Animals Schedule Doses 1 10 males
Vehicle Control Day -1 to Day 29 31 2 10 males SCV-07 100 .mu.g/kg
Day -1 to Day 29 31 3 10 males SCV-07 100 .mu.g/kg Day 0 to Day 3 8
and Day 6 to Day 9 4 10 males SCV-07 100 .mu.g/kg Day -1, Days 4, 5
23 Day 10 to Day 29 Injections of SCV-07 on Days 0, 1, 2, 3, 6, 7,
8 and 9 were given approximately 30 minutes prior to radiation.
5. Material and Methods
5.1 Location of Study Performance
[0094] The study was performed at Biomodels AAALAC-accredited
facility in Watertown, Mass.
5.2 Animals
[0095] Male LVG Syrian Golden Hamsters (Charles River
Laboratories), aged 5 to 6 weeks, with average body weight of 81.0
g at study commencement, were used. Animals were individually
numbered using an ear punch and housed in small groups of
approximately 10 animals per cage. Animals were acclimatized for 5
days prior to study commencement and during this period, the
animals were observed daily in order to reject animals that present
in poor condition.
5.3 Housing
[0096] 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. Bedding was
changed a minimum of once per week. Cages, tops, bottles, etc. were
washed with a commercial detergent and allowed to air dry. 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 marked all cages. The temperature and relative
humidity was recorded during the study, and the records
retained.
5.4 Diet
[0097] Animals were fed with a Purina Labdiet.RTM. 5061 rodent diet
and water was provided ad libitum.
5.5 Animal Randomization and Allocations.
[0098] Animals 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.
For more consistent identification, ear punch numbering was used
rather than tagging, since tags may become dislodged during the
course of the study. A cage card was used to identify each cage or
label marked with the study number (SCI-03), treatment group number
and animal numbers.
5.6 Sub-Cutaneous Dosing and Drug Application
[0099] The test compound, human SCV-07 peptide was provided as a
powder and dissolved in sterile PBS immediately prior to
administration. Drug was given in a volume of 0.1, using a
tuberculin syringe with a 27G needle. Injections were given
subcutaneously to the back or abdomen.
5.7 Mucositis Induction
[0100] Radiation was generated with a Philips 160 kVp (kilovolt
potential) (18.75-ma) X-ray source at a focal distance of 30 cm,
with a 3.0 mm hardened Al filtration system. Irradiation was
targeted to the left buccal pouch mucosa at a rate of 3.32
Gy/minute. Calibration of this source with a Victoreen model 530
dosimeter indicated that the dose rate was 28.57 nC/min. Using this
calibration, the energy received by each animal at each radiation
dose was approximately 64.5 nC (nanoCoulombs) at each time point.
Prior to irradiation, animals were anesthetized with an
intraperitoneal injection of ketamine (160 mg/kg) and xylazine (8
mg/kg). The left buccal pouch was everted, fixed and isolated using
a lead shield.
5.8 Mucositis Scoring
[0101] The mucositis score, weight change and survival were
measured throughout the study as described above. For the
evaluation of mucositis, the animals were anesthetized with an
inhalation anesthetic, and the left pouch everted. Mucositis was
scored visually by comparison to a validated photographic scale,
ranging from 0 for normal, to 5 for severe ulceration (clinical
scoring). In descriptive terms, this scale is defined as
follows:
TABLE-US-00014 TABLE 10 SCI-03: Mucositis Scoring. Score:
Description: 0 Pouch completely healthy. No erythema or
vasodilation. 1 Light to severe erythema and vasodilation. No
erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of
superficial aspects of mucosa leaving denuded areas, but no frank
ulceration. Decreased stippling of mucosa. 3 Formation of off-white
ulcers in one or more places. Ulcers may have a yellow/gray due to
pseudomembrane. Cumulative size of ulcers should equal about 1/4 of
the pouch. Severe erythema and vasodilation. 4 Cumulative seize of
ulcers should equal about 1/2 of the pouch. Loss of pliability.
Severe erythema and vasodilation. 5 Virtually all of pouch is
ulcerated. Loss of pliability (pouch can only partially be
extracted from mouth).
[0102] A score of 1-2 is considered to represent a mild stage of
the disease, whereas a score of 3-5 is considered to indicate
moderate to severe mucositis. Following visual scoring, a
photograph was taken of each animal's mucosa using a standardized
technique. At the conclusion of the experiment, all films were
developed and the photographs randomly numbered. At least two
independent trained observers graded the photographs in blinded
fashion using the above-described scale (blinded scoring).
6. Results
6.1 Survival
[0103] One death occurred during this study in the control group on
day 8 as a consequence of anesthesia for radiation.
6.2 Weight Change
[0104] There were no significant differences in weight changes
between study groups. The mean daily percent weight change data was
evaluated. The saline treated control hamsters gained an average of
76.3% of their starting weight during the study. Hamsters in the
group receiving SCV-07 at 100 .mu.g/kg on days -1 to 29 gained an
average of 80.7% of their starting weight during the study.
Hamsters in the group receiving SCV-07 at 100 .mu.g/kg on days of
radiation only gained an average of 66.3% of their starting weight
during the study. Hamsters in the group receiving SCV-07 at 100
.mu.g/kg on day -1, day 4, day 5 and days 10-29 gained an average
of 69.7% of their starting weight during the study. The
significance of these differences was evaluated by calculating the
area-under-the-curve (AUC) for the weight gain of each animal, and
then comparing the different treatment groups using a One-Way ANOVA
test. The results of this analysis indicated that there were no
significant differences between the different treatment groups
(P=0.490).
6.3 Mucositis (Tables 11 & 12)
[0105] The kinetics and severity of mucositis development among
control animals was consistent with that which was expected.
[0106] Mean daily mucositis scores for each group were evaluated.
In the saline treated control group, the mean peak mucositis score
was 3.2, which occurred on day 19. The group receiving SCV-07 from
day -1 to day 29 had a peak mean mucositis score of 3.3, which
occurred on day 19. The group receiving SCV-07 on the days of
radiation (0-3 and 6-9) had a peak mean mucositis score of 3.0,
which occurred on day 17. The group receiving SCV-07 on days 1, 4,
5 and 10 through 29 had a peak mean mucositis score of 2.9, which
occurred on days 17, 19 and 23. The significance of the differences
observed between the different treatment groups was evaluated by
calculating the number of days with a score of 3 or higher for each
group and comparing these numbers using a chi-squared
(.quadrature..sup.2) test. The results of this analysis are shown
in Table 11. The hamsters in the saline treated control group had a
score of 3 or higher on 36% of the animal days evaluated. In the
group receiving SCV-07 from day -1 to day 29, a mucositis score of
3 or higher was observed on 32.7% of the animals days evaluated,
which was not statistically significantly different from controls
(P=0.448). In the group receiving SCV-07 on days 0-3 and days 6-9,
a mucositis score of 3 or higher was observed on 24% of the animals
days evaluated, which was statistically significantly different
from controls (P=0.002). In the group receiving SCV-07 on days -1,
day 4 day 5 and day 10 to day 29, a mucositis score of 3 or higher
was observed on 30.7% of the animals days evaluated, which was not
statistically significantly different from controls (P=0.204). A
further analysis of the mucositis scores was performed using the
Mann-Whitney rank sum analysis to compare the scores for each group
on each day. The results of this analysis are shown in Table 12. In
this analysis, 2 days of significant reduction in the mucositis
score are generally required before it is regarded as meaningful.
The group treated with SCV-07 on days -1 to 29 was significantly
better than the saline controls on days 29 (P=0.004), 31 (P=0.017)
and 33 (P=0.002). The group treated with SCV-07 on days 0 to 3 and
day 6 to day 9 was significantly better than the saline controls on
days 21 (P=0.047), 23 (P<0.001), 25 (P=0.009), 29 (P<0.001),
31 (P=0.015) and 33 (P<0.001). The group treated with SCV-07 on
days -1, 4, 5 and days 10 to 29 was significantly better than the
saline controls on days 29 (P<0.001), 31 (P=0.004) and 33
(P<0.001).
TABLE-US-00015 TABLE 11 SCI-03. Chi-square analysis of the total
number of days the animals in each group spent with a score of
three or more. This statistic is a measure of severity of
ulceration, a clinically important outcome. Days Days Total % Days
Chi Sq Group >=3 <3 Days >=3 v control P Value Vehicle
(PBS), qd, 98 174 272 36.0 -- -- sc Days -1 to 29 SCV-07 98 202 300
32.7 0.575 0.448 100 ug/kg, qd, sc Days -1 to 29 SCV-07 72 228 300
24.0 0.3160 0.002 100 ug/kg, qd, sc Days 0 to 3 and 6 to 9 SCV-07
92 208 300 30.7 0.2300 0.204 100 ug/kg, qd, sc Days -1, 4, 5 10 to
29
TABLE-US-00016 TABLE 12 SCI-03. The significance of group
differences observed in daily mucositis scores was determined using
the Mann-Whitney rank sum test. This nonparametric statistic is
appropriate for the visual mucositis scoring scale. The p values
for each calculation are shown. Significant reductions in mucositis
scores relative to controls are shown underlined. Day Group
Comparison 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Control vs
0.989 0.295 0.526 0.297 0.490 0.988 0.608 0.129 0.062 0.781 0.526
0.004 0.017 0.002 0.872 SCV-7 100 ug/kg, qd, sc Days -1 to 29
Control vs 0.989 0.296 0.244 0.080 0.490 0.988 0.115 0.047
<0.001 0.009 0.224 <0.001 0.015 <0.001 0.872 SCV-7 100
ug/kg, qd, sc Days 0 to 3 and 6 to 9 Control vs 0.989 0.605 0.918
0.406 0.164 0.605 0.115 0.129 0.648 0.420 0.528 <0.001 0.004
<0.001 0.490 SCV-7 100 ug/kg, qd, sc Days -1, 4, 5 10 to 29
7. Conclusions
[0107] 1. There was no evidence of any toxicity from SCV-07 in this
study based on the observations of mortality and weight gain. 2.
Animals treated with human SCV-07 at 100 .mu.g/kg on days on which
radiation was administered (days 0-3 and 6-9) showed a
statistically significant reduction in the number of days with a
mucositis score of 3 or higher (P=0.002), and a significant
reduction in mucositis scores on days 21 (P=0.047), 23
(P<0.001), 25 (P=0.009), 29 (P<0.001), 31 (P=0.015) and 33
(P<0.001). This result suggests that SCV-07 may be effective in
reducing the overall severity of mucositis, which, consequently,
results in enhanced resolution of mucosal injury. 3. Hamsters
treated with SCV-07 on days -1 to 29 or on days on which radiation
was not administered (days -1, 4, 5 and 10-29), did not show a
significant reduction in the number of animal days with a score of
3 or higher, but did show significant reductions in mucositis
scores on days 29, 31 and 33. 4. The contrast in effect of SCV-07
based on the schedule of radiation administration may provide some
insight into its mechanism of action, but requires further
evaluation. The fact that SCV-07 schedules in which the drug was
administered on non-radiation days were not as efficacious is of
interest, especially for animals treated on consecutive days.
Furthermore, the observation that all animals treated with SCV-07
responded identically at the late stages of the study might suggest
multiple effects of SCV-07 on the overall pathogenesis and
resolution of radiation-induced mucositis.
9. Appendices
TABLE-US-00017 [0108] APPENDIX 5 Animal Weights Day Group Animal -1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 1 82 87 86 88 89 89
94 95 98 97 100.8 103 105 108 109 110 110 112 115 1 2 78 82 81 84
87 85 91 94 95 Dead 1 3 79 79 77 76 75 75 77 77 79 80 82.2 85 85 89
88 87 86 86 89 1 4 88 92 93 96 98 99 103 107 108 107 113.9 116 116
120 121 123 122 124 125 1 5 77 80 79 84 88 86 91 93 90 88 96 96 96
97 98 98 98 100 104 1 6 83 85 90 91 95 94 90 100 100 101 106.1 107
109 112 113 114 115 115 118 1 7 76 78 78 79 81 83 98 88 90 89 93.4
95 93 96 98 98 99 99 102 1 8 73 76 75 76 77 78 84 86 86 87 90.5 93
97 98 99 101 103 102 103 1 9 81 86 87 88 87 90 94 99 98 100 104.4
105 108 111 113 113 114 114 119 1 10 79 83 84 82 84 80 88 90 91 90
93.7 95 95 97 96 97 95 100 102 2 11 82 86 89 78 77 80 83 88 88 89
94.5 95 98 100 103 104 104 105 108 2 12 80 82 82 83 86 85 88 92 91
90 93.4 92 95 96 97 96 97 99 100 2 13 82 84 84 87 88 88 92 94 93 94
98.3 100 99 103 104 106 105 107 108 2 14 73 77 76 79 79 82 84 87 87
87 90.6 92 96 97 97 98 99 99 101 2 15 82 87 89 92 95 95 98 102 102
103 108.5 108 110 114 115 116 117 117 120 2 16 83 85 88 91 94 93 97
102 99 99 103.5 105 107 111 111 109 108 109 111 2 17 81 82 83 87 88
87 91 95 93 95 99.7 97 101 104 106 105 107 106 107 2 18 76 77 76 80
79 79 84 86 85 87 88.2 92 92 95 94 95 94 96 97 2 19 84 87 87 88 90
91 94 98 100 98 102.3 104 104 107 108 108 108 111 110 2 20 69 70 74
76 75 76 79 79 84 84 88.6 90 94 97 98 98 98 99 103 3 21 87 92 92 97
94 95 100 104 106 103 110.6 111 112 102 114 113 112 113 118 3 22 76
78 79 80 80 82 85 89 90 88 94.6 95 96 110 101 100 100 103 106 3 23
93 98 95 102 104 102 108 111 115 113 117.4 119 122 125 124 125 126
129 132 3 24 78 82 79 80 82 81 85 88 89 88 92.6 93 95 97 98 99 97
97 100 3 25 80 84 82 85 84 86 88 90 91 93 94.4 96 99 101 103 100
102 101 104 3 26 87 89 86 88 86 86 89 91 89 90 93.7 95 97 98 101
100 101 100 101 3 27 84 87 87 88 89 88 93 97 97 97 101.7 103 104
103 109 112 111 113 115 3 28 80 82 83 84 88 85 88 94 93 94 97.5 98
99 99 105 103 105 106 106 3 29 79 79 78 81 83 83 87 89 90 88 91.6
94 94 96 96 97 96 99 102 3 30 76 76 73 74 76 74 77 78 78 78 79.2 80
81 84 84 84 82 85 86 4 31 80 84 85 89 90 93 94 96 97 98 101.2 102
104 105 107 105 106 109 111 4 32 85 88 90 91 94 94 99 102 103 105
110.3 110 112 116 116 116 116 117 117 4 33 82 85 82 96 87 85 89 92
93 91 95.3 95 96 97 97 96 96 96 96 4 34 87 89 87 90 86 91 97 99 101
103 108.6 110 110 115 117 118 120 122 123 4 35 84 89 90 92 97 97
104 107 110 108 111.7 114 115 117 119 121 122 124 127 4 36 85 90 89
91 94 96 98 101 103 102 104.7 108 107 109 110 110 111 112 113 4 37
87 91 91 92 94 95 98 101 102 102 106.7 108 109 112 113 113 113 113
114 4 38 89 94 92 97 98 99 106 106 109 106 110.6 112 114 115 117
117 119 121 122 4 39 70 73 72 73 73 73 76 79 78 79 80.7 80 78 82 81
83 83 86 87 4 40 81 87 86 85 88 87 87 90 92 89 90.6 89 90 93 88 88
89 94 97 Day Group Animal 18 19 20 21 22 23 24 25 26 27 28 29 30 31
32 33 34 35 1 1 115 117 119 120 122 124 125 126 130 130 132 132 135
137 137 141 143 144 1 2 Dead 1 3 90 95 95 97 98 101 101 102 106 108
107 108 111 114 115 119 118 120 1 4 126 129 130 128 130 133 131 131
134 135 138 136 138 138 139 143 142 143 1 5 105 106 109 113 114 117
120 121 126 129 129 128 134 135 137 142 143 142 1 6 120 123 126 128
130 132 138 138 144 147 146 147 152 155 155 160 159 159 1 7 103 109
111 111 111 116 118 118 124 123 125 125 130 131 133 137 138 137 1 8
103 110 109 110 112 115 115 117 121 122 123 121 126 129 129 133 131
134 1 9 120 124 128 126 129 132 132 134 138 139 140 140 145 146 147
151 155 153 1 10 105 105 109 110 110 113 114 115 118 119 121 120
122 124 125 129 131 133 2 11 108 113 114 116 117 120 122 123 129
131 134 132 136 138 141 146 145 146 2 12 98 104 105 107 108 111 113
113 117 116 118 118 122 124 126 131 130 131 2 13 109 114 116 117
119 120 124 124 129 130 130 130 137 138 141 136 145 136 2 14 103
107 109 110 112 114 116 117 121 124 124 123 128 131 120 145 136 145
2 15 120 124 126 127 127 130 132 132 137 138 139 139 142 143 143
147 146 147 2 16 113 116 117 122 123 126 128 128 132 133 135 134
138 142 142 148 147 148 2 17 118 122 121 126 127 130 130 132 136
137 140 140 144 146 147 152 153 154 2 18 98 102 103 104 105 108 111
110 114 115 114 114 120 121 122 125 125 127 2 19 113 115 116 119
120 120 123 126 129 130 133 133 137 139 139 144 147 146 2 20 106
108 111 118 117 118 120 121 124 126 131 127 133 138 138 144 146 147
3 21 117 122 123 124 127 127 128 128 131 133 134 132 135 138 139
143 143 144 3 22 109 112 114 115 114 121 122 121 124 125 127 126
130 132 133 136 136 137 3 23 137 138 141 141 144 152 148 150 153
158 158 158 162 165 165 172 171 170 3 24 101 105 107 108 108 113
114 115 119 119 122 122 126 128 128 133 134 135 3 25 104 108 109
108 112 113 115 116 117 119 121 120 122 123 124 127 127 128 3 26
102 106 108 109 111 113 115 116 122 119 121 122 125 128 130 133 133
135 3 27 108 111 112 114 115 118 120 121 124 124 125 126 129 131
131 132 134 134 3 28 108 113 114 114 118 120 121 122 120 126 128
128 132 134 134 140 138 140 3 29 104 107 107 109 111 114 115 114
119 122 120 121 125 125 128 131 131 132 3 30 87 90 90 93 93 96 95
95 97 97 100 99 102 104 103 107 107 110 4 31 112 118 118 120 124
124 123 125 126 129 131 131 133 136 138 141 140 141 4 32 114 120
121 124 124 127 128 131 134 134 136 134 138 142 142 146 147 146 4
33 95 98 99 101 102 105 107 108 110 114 114 112 116 118 119 122 122
124 4 34 122 128 128 131 133 135 138 139 142 143 145 146 150 152
153 158 157 158 4 35 127 132 133 136 134 137 138 140 143 144 146
144 149 151 152 157 158 159 4 36 115 119 118 120 122 123 124 128
128 131 131 131 136 136 138 141 140 141 4 37 113 118 118 119 120
120 123 122 123 127 128 128 132 134 134 137 139 140 4 38 121 127
128 131 133 135 135 137 139 140 142 142 146 150 149 154 153 154 4
39 88 92 92 94 95 97 99 98 100 102 103 102 105 108 109 112 112 113
4 40 98 102 104 109 109 111 112 113 118 121 122 118 126 126 129 132
133 134
TABLE-US-00018 APPENDIX 6 Mucositis Scores Day Number Group Animal
7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 1 1 0 1 1 1 2 3 4 3 3 3
3 2 2 2 1 1 1 0 1 1 1 2 3 4 3 3 3 3 2 2 2 1 1 2 0 dead 1 2 0 1 3 0
1 1 2 3 3 4 3 3 2 2 2 2 2 1 1 3 0 1 1 2 3 3 4 3 3 2 2 2 2 2 1 1 4 0
1 1 2 3 3 3 3 3 2 2 1 1 2 1 1 4 0 1 1 2 3 3 3 3 3 2 2 1 1 2 1 1 5 0
1 2 2 3 3 3 3 2 2 2 2 2 2 1 1 5 0 1 2 2 3 3 3 3 2 2 2 2 2 2 1 1 6 0
1 1 2 3 3 3 3 3 3 2 2 2 2 1 1 6 0 1 1 2 3 3 3 3 3 3 2 2 2 2 1 1 7 0
1 1 2 3 3 3 3 3 2 2 1 1 1 0 1 7 0 1 1 2 3 3 3 3 3 2 2 1 1 1 0 1 8 0
1 1 1 2 3 3 4 4 3 2 2 1 1 0 1 8 0 1 1 1 2 3 3 4 4 3 2 2 1 1 0 1 9 0
1 1 1 2 3 3 3 3 2 2 1 0 1 0 1 9 0 1 1 1 2 3 3 3 3 2 2 1 0 1 0 1 10
0 1 2 2 3 3 3 3 3 3 3 3 3 2 1 1 10 0 1 2 2 3 3 3 3 3 3 3 3 3 2 1 2
11 0 1 1 2 3 3 3 3 3 3 3 3 2 2 1 2 11 0 1 1 2 3 3 3 3 3 3 3 3 2 2 1
2 12 0 0 1 3 3 3 3 3 3 2 2 1 0 1 0 2 12 0 0 1 3 3 3 3 3 3 2 2 1 0 1
0 2 13 0 1 1 2 3 3 4 3 2 2 2 1 1 1 0 2 13 0 1 1 2 3 3 4 3 2 2 2 1 1
1 0 2 14 0 1 1 2 3 3 4 3 3 3 2 1 1 1 1 2 14 0 1 1 2 3 3 4 3 3 3 2 1
1 1 1 2 15 0 1 1 2 2 3 3 3 2 3 2 1 1 1 1 2 15 0 1 1 2 2 3 3 3 2 3 2
1 1 1 1 2 16 0 1 1 2 3 3 4 3 3 3 2 1 1 1 1 2 16 0 1 1 2 3 3 4 3 3 3
2 1 1 1 1 2 17 0 1 1 1 3 3 2 2 3 2 2 1 0 0 0 2 17 0 1 1 1 3 3 2 2 3
2 2 1 0 0 0 2 18 0 1 1 1 2 3 3 3 2 3 2 0 1 1 1 2 18 0 1 1 1 2 3 3 3
2 3 2 0 1 1 1 2 19 0 0 2 2 3 3 3 3 3 2 2 1 1 1 1 2 19 0 0 2 2 3 3 3
3 3 2 2 1 1 1 1 2 20 0 1 1 2 3 3 4 2 2 2 2 1 1 1 1 2 20 0 1 1 2 3 3
4 2 2 2 2 1 1 1 1 3 21 0 1 1 2 3 3 3 3 2 2 2 1 2 1 1 3 21 0 1 1 2 3
3 3 3 2 2 2 1 2 1 1 3 22 0 2 1 2 3 3 3 2 2 2 2 0 0 0 0 3 22 0 2 1 2
3 3 3 2 2 2 2 0 0 0 0 3 23 0 0 1 2 3 3 3 2 2 2 2 1 0 1 1 3 23 0 0 1
2 3 3 3 2 2 2 2 1 0 1 1 3 24 0 1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 24 0 1
1 2 3 3 3 3 2 2 2 1 1 1 1 3 25 0 1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 25 0
1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 26 0 0 1 2 2 3 3 3 2 2 2 1 1 1 1 3 26
0 0 1 2 2 3 3 3 2 2 2 1 1 1 1 3 27 0 1 1 2 3 3 3 3 3 2 2 1 1 1 1 3
27 0 1 1 2 3 3 3 3 3 2 2 1 1 1 1 3 28 0 1 1 2 3 3 3 3 3 2 2 1 0 1 0
3 28 0 1 1 2 3 3 3 3 3 2 2 1 0 1 0 3 29 0 0 1 2 3 3 3 3 2 2 2 1 2 1
1 3 29 0 0 1 2 3 3 3 3 2 2 2 1 2 1 1 3 30 0 1 1 2 2 3 2 2 2 1 2 0 0
0 0 3 30 0 1 1 2 2 3 2 2 2 1 2 0 0 0 0 4 31 0 0 1 2 3 3 3 3 3 2 2 1
0 1 0 4 31 0 0 1 2 3 3 3 3 3 2 2 1 0 1 0 4 32 0 1 1 2 2 3 3 2 2 3 2
0 1 1 1 4 32 0 1 1 2 2 3 3 2 2 3 2 0 1 1 1 4 33 0 1 1 2 2 3 3 3 3 2
2 0 1 1 1 4 33 0 1 1 2 2 3 3 3 3 2 2 0 1 1 1 4 34 0 1 1 1 2 2 3 3 3
3 2 2 2 2 1 4 34 0 1 1 1 2 2 3 3 3 3 2 2 2 2 1 4 35 0 1 2 2 3 3 3 3
3 3 2 1 0 1 1 4 35 0 1 2 2 3 3 3 3 3 3 2 1 0 1 1 4 36 0 1 1 2 2 3 3
3 3 3 2 1 1 1 1 4 36 0 1 1 2 2 3 3 3 3 3 2 1 1 1 1 4 37 0 1 1 1 2 3
3 3 3 3 2 0 1 1 1 4 37 0 1 1 1 2 3 3 3 3 3 2 0 1 1 1 4 38 0 1 1 2 2
3 3 3 3 3 3 1 1 1 1 4 38 0 1 1 2 2 3 3 3 3 3 3 1 1 1 1 4 39 0 1 1 2
3 3 3 3 3 2 2 1 0 0 0 4 39 0 1 1 2 3 3 3 3 3 2 2 1 0 0 0 4 40 0 1 2
2 3 3 2 2 3 2 2 1 0 0 0 4 40 0 1 2 2 3 3 2 2 3 2 2 1 0 0 0
Example 4
A Study of SCV-07 in the Treatment of Oral Mucositis Induced by a
Combination of Cisplatin and Acute Radiation in Hamsters.
Introduction
[0109] Prior studies with SCV-07 have shown that it is effective in
treating oral mucositis in hamster models of the disease induced by
both acute and fractionated radiation. In this study, the efficacy
of SCV-07 was evaluated in a hamster model of oral mucositis
induced by a combination of chemotherapy and radiation, and
specifically, by the combination of cis-platin and radiation.
Methods
[0110] Forty Golden Syrian hamsters were prospectively randomized
into four equally sized groups. Mucositis was induced on the left
cheek pouch mucosa of golden Syrian hamsters by a single dose of
cis-platin at 5 mg/kg given on day -1, and a single dose of
radiation administered on day 0 at a dose of 35Gy. Beginning on day
-1 and continuing once daily until day 20, SCV-07 was given by
sub-cutaneous injection at doses of 10 .mu.g/kg, 100 .mu.g/kg or 1
mg/kg in a volume of 100 .mu.L. Animals' activity and weight were
evaluated daily. Beginning on day 6 and continuing on alternate
days for the duration of the study, oral mucositis was evaluated
using a standard scoring six point scale. The number of days of
ulcerative mucositis was evaluated using a Chi-squared test of
scores of .gtoreq.3 throughout the study, and the individual daily
group scores were assessed with a Rank Sum Test.
Results
[0111] Mucositis was favorably and consistently impacted in animals
treated with SCV-07 in all test groups. Severe mucositis was
reduced from 50% of animals days evaluated in vehicle controls to
approximately 30% in SCV-07 treated animals. The most significant
impact was seen in the later stages of the disease process, after
the peak of mucositis. No significant differences were observed
among groups relative to weight changes. No animals died during the
course of the experiment.
Conclusions
[0112] 1. There was no evidence of any toxicity from SCV-07 in this
study based on the observations of mortality and weight gain. 2.
Animals treated with SCV-07 at 10 .mu.g/kg from day -1 to day 20
showed a statistically significant reduction in the number of days
with a mucositis score of 3 or higher (P<0.001), and a
significant reduction in mucositis scores on days 22 (P=0.010), 24
(P=0.022), 26 (P=0.015), and 28 (P<0.001). 3. Animals treated
with SCV-07 at 100 .mu.g/kg from day -1 to day 20 showed a
statistically significant reduction in the number of days with a
mucositis score of 3 or higher (P<0.001), and a significant
reduction in mucositis scores on days 6 (P=0.015), 22 (P=0.003), 24
(P=0.005), 26 (P<0.001), and 28 (P<0.001). 4. Animals treated
with SCV-07 at 1 mg/kg from day -1 to day 20 showed a statistically
significant reduction in the number of days with a mucositis score
of 3 or higher (P<0.001), and a significant reduction in
mucositis scores on days 6 (P=0.015), 20 (P=0.029), 22 (P=0.012),
24 (P=0.001), 26 (P=0.003), and 28 (P<0.001).
1.1 Background
[0113] KGF-1 and other FGF family members have been shown to induce
epithelial thickening of the oral and esophageal mucosal surfaces
in BDF-1. SCV-07 and the derived SCV-07 peptide are believed to
have mechanisms that that may overlap with KGF-1, and have been
shown to be protective in other models of mucosal injury.
[0114] Oral ulcerative mucositis is a common, painful,
dose-limiting toxicity of drug and radiation therapy for cancer.
The disorder is characterized by breakdown of the oral mucosa that
results in the formation of ulcerative lesions. In granulocytopenic
patients, the ulcerations that accompany mucositis are frequent
portals of entry for indigenous oral bacteria often leading to
sepsis or bacteremia. Mucositis occurs to some degree in more than
one third of patients receiving anti-neoplastic drug therapy. The
frequency and severity are significantly greater among patients who
are treated with induction therapy for leukemia or with many of the
conditioning regimens for bone marrow transplant. Among these
individuals, moderate to severe mucositis is not unusual in more
than three-quarters of patients. Moderate to severe mucositis
occurs in virtually all patients who receive radiation therapy for
tumors of the head and neck and typically begins with cumulative
exposures of 15 Gy and then worsens as total doses of 60 Gy or more
are reached.
[0115] Clinically mucositis progresses through three stages:
1. Painful erythema which can generally be managed by topical
anesthetics or non-narcotic analgesics. 2. Painful ulceration often
with pseudomembrane formation. In the case of concomitant
myelosuppression, bacteremias or sepsis of oral origin are not
uncommon. Pain is often of such intensity as to require narcotic
analgesia, frequently parenterally. 3. Spontaneous healing,
occurring about 2-3 weeks after cessation of anti-neoplastic
therapy.
[0116] Currently, the only approved biologic or drug for mucositis
prevention and/or treatment is Kepivance (palifermin). Kepivance
use is limited to mucositis in patients receiving stem cell
transplant for hematologic malignancies. Consequently, standard
therapy for mucositis consists of palliative rinses, such as
saline, bicarbonate solutions, mouthwashes, topical analgesics such
as lidocaine and/or systemic administration of narcotics.
[0117] The complexity of mucositis as a biological process has only
been recently appreciated. It has been suggested that the condition
represents a sequential interaction of oral mucosal cells and
tissues reactive oxygen species, pro-inflammatory cytokines,
mediators of apoptosis, a range of signaling pathways, and local
factors such as saliva and the oral micro biota. While epithelial
degeneration and breakdown ultimately result in mucosal ulceration,
it appears that the early changes associated with radiation-induced
mucosal toxicity occur within the endothelium, and connective
tissue of the submucosa. It appears that the overall mechanism for
mucositis development is similar for both radiation and
chemotherapy.
1.2 Chemo-Radiation Model of Oral Mucositis with Cis-Platin.
[0118] The chemo-radiation model of oral mucositis in hamsters,
developed by the Principal Investigator, has proven to be an
accurate, efficient and cost-effective technique to provide a
preliminary evaluation of anti-mucositis compounds. In this model,
hamsters received a single dose of cis-platin at 5 mg/kg on day -1,
followed by a single dose of radiation of 35 Gy to the left cheek
pouch on day 0, rather than the single dose of 40 Gy on day 0 that
is used in acute radiation studies. The course of mucositis in this
model is very similar to the acute radiation model and results in
peak mucositis scores approximately 16-18 days following
radiation.
2. Study Objective and Summary
2.1 Study Objective
[0119] The objective of this study was to evaluate the effect of
SCV-07, administered by sub-cutaneous injection, on the frequency,
severity and duration of oral mucositis induced by a fractionated
radiation protocol.
2.2 Study Summary
[0120] Hamsters received a single dose of cis-platin at 5 mg/kg on
day -1 by intraperitoneal injection, followed by a single dose of
radiation of 35 Gy to the left cheek pouch on day 0. This was
accomplished by anesthetizing the animals and everting the left
buccal pouch, while protecting the rest of the animal's bodies with
a lead shield. Test materials were given by sub-cutaneous injection
once daily as detailed in Table 13. Mucositis was evaluated
clinically starting on day 6, and continuing on alternate days
until day 28. Test articles were given as from day -1 to day
20.
3. Evaluation
3.1 Mucositis Evaluation
[0121] The grade of mucositis was scored, beginning day 6, and for
every second day thereafter, through and including day 28. The
effect on mucositis of each drug treatment compared to placebo was
assessed according to the following parameters:
3.1.1 The Difference in the Number of Days Hamsters in Each Group
have Ulcerative (Score .gtoreq.3) Mucositis.
[0122] On each evaluation day, the number of animals with a blinded
mucositis score of .gtoreq.3 in each drug treatment group was
compared to the control group. Differences were compared on a
cumulative basis and statistical significance was determined by
chi-square analysis. Efficacy, in this analysis, is defined by a
significant reduction in the number of days that a group of animals
had ulcerations (scores .gtoreq.3) when compared to the control
group.
3.1.2 Rank Sum Differences in Daily Mucositis Scores.
[0123] For each evaluation day the scores of the control group were
compared to those of the treated groups using non-parametric rank
sum analysis. Treatment success was considered as a statistically
significant lowering of scores in the treated group on 2 or more
days from day 6 to day 28.
3.2 Weights and Survival
[0124] All animals were weighed daily and their survival recorded,
in order to assess possible differences in animal weight among
treatment groups as an indication for mucositis severity and/or
possible toxicity resulting from the treatments.
4. Study Design
[0125] Forty (40) male Syrian Golden Hamsters were given an
intraperitoneal injection of 5 mg/kg Cisplatin on day -1. On day 0
all animals were given an acute radiation dose of 35 Gy directed to
their left buccal cheek pouch. This was accomplished by
anesthetizing the animals and everting the left buccal pouch, while
protecting the rest of the animals with a lead shield. Test
materials were given by subcutaneous injection once daily as
detailed in Table 13. Mucositis was evaluated clinically starting
on Day 6, and continued on alternate days until day 28.
TABLE-US-00019 TABLE 13 SCI-04. Study Design Number of Treatment
Volume Group Animals Cisplatin Radiation Treatment Schedule* (mL) 1
10 males 5 mg/kg, 35 Gy, day 0 Vehicle (PBS), sc, qd Day -1 to 20
Adjust per day -1 body weight 2 10 males 5 mg/kg, 35 Gy, day 0
SCV-07, sc, qd Day -1 to 20 Adjust per day -1 10 .mu.g/kg, body
weight 3 10 males 5 mg/kg, 35 Gy, day 0 SCV-07, sc, qd Day -1 to 20
Adjust per day -1 100 .mu.g/kg, body weight 4 10 males 5 mg/kg, 35
Gy, day 0 SCV-07, sc, qd Day -1 to 20 Adjust per day -1 1.0 mg/kg
body weight *The dose on day 0 will be performed 30 minutes prior
to radiation
5. Material and Methods
5.1 Location of Study Performance
[0126] The study was performed at Biomodels AAALAC-accredited
facility in Watertown, Mass. IACUC approval number 07-0620-01 for
this study was obtained from Biomodels IACUC.
5.2 Animals
[0127] Male LVG Syrian Golden Hamsters (Charles River
Laboratories), aged 5 to 6 weeks, with average body weight of 90 g
at study commencement, were used. Animals were individually
numbered using an ear punch and housed in small groups of
approximately 10 animals per cage. Animals were acclimatized for 5
days prior to study commencement and during this period, the
animals were observed daily in order to reject animals that present
in poor condition.
5.3 Housing
[0128] 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. Bedding was
changed a minimum of once per week. Cages, tops, bottles, etc. were
washed with a commercial detergent and allowed to air dry. 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 marked all cages. The temperature and relative
humidity was recorded during the study, and the records
retained.
5.4 Diet
[0129] Animals were fed with a Purina Labdiet.RTM. 5061 rodent diet
and water was provided ad libitum.
5.5 Animal Randomization and Allocations.
[0130] Animals 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.
For more consistent identification, ear punch numbering was used
rather than tagging, since tags may become dislodged during the
course of the study. A cage card was used to identify each cage or
label marked with the study number (SCI-04), treatment group number
and animal numbers.
5.6 Sub-Cutaneous Dosing and Drug Application
[0131] The test compound, human SCV-07 peptide was provided as a
powder and dissolved in sterile PBS immediately prior to
administration. Drug was given in a volume of 0.1, using a
tuberculin syringe with a 27G needle. Injections were given
subcutaneously to the back or abdomen.
5.7 Mucositis Induction
[0132] In this study, mucositis was induced with a combination of
Cis-platin and radiation. Cis-platin was given as a single
injection (IP) of 5 mg/kg on day -1. Radiation was given as a
single focal dose of 35 Gy on day 0. Radiation was generated with a
Philips 160 kVp (kilovolt potential) (18.75-ma) X-ray source at a
focal distance of 30 cm, with a 3.0 mm hardened Al filtration
system. Irradiation was targeted to the left buccal pouch mucosa at
a rate of 3.32 Gy/minute. Calibration of this source with a
Victoreen model 530 dosimeter indicated that the dose rate was
28.57 nC/min. Using this calibration, the energy received by each
animal at each radiation dose was approximately 688.6 nC
(nanoCoulombs). Prior to irradiation, animals were anesthetized
with an intraperitoneal injection of ketamine (160 mg/kg) and
xylazine (8 mg/kg). The left buccal pouch was everted, fixed and
isolated using a lead shield.
5.8 Mucositis Scoring
[0133] The mucositis score, weight change and survival were
measured throughout the study as described above. For the
evaluation of mucositis, the animals were anesthetized with an
inhalation anesthetic, and the left pouch everted. Mucositis was
scored visually by comparison to a validated photographic scale,
ranging from 0 for normal, to 5 for severe ulceration (clinical
scoring). In descriptive terms, this scale is defined as
follows:
TABLE-US-00020 TABLE 14 SCI-04: Mucositis Scoring. Score:
Description: 0 Pouch completely healthy. No erythema or
vasodilation. 1 Light to severe erythema and vasodilation. No
erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of
superficial aspects of mucosa leaving denuded areas, but no frank
ulceration. Decreased stippling of mucosa. 3 Formation of off-white
ulcers in one or more places. Ulcers may have a yellow/gray due to
pseudomembrane. Cumulative size of ulcers should equal about 1/4 of
the pouch. Severe erythema and vasodilation. 4 Cumulative seize of
ulcers should equal about 1/2 of the pouch. Loss of pliability.
Severe erythema and vasodilation. 5 Virtually all of pouch is
ulcerated. Loss of pliability (pouch can only partially be
extracted from mouth).
[0134] A score of 1-2 is considered to represent a mild stage of
the disease, whereas a score of 3-5 is considered to indicate
moderate to severe mucositis. Following visual scoring, a
photograph was taken of each animal's mucosa using a standardized
technique. At the conclusion of the experiment, all films were
developed and the photographs randomly numbered. At least two
independent trained observers graded the photographs in blinded
fashion using the above-described scale (blinded scoring).
6. Results and discussion
6.1 Survival
[0135] No deaths were seen in this study
6.2 Weight Change
[0136] There were no significant differences in weight changes
between study groups. The mean daily percent weight change data was
evaluated. The saline treated control hamsters gained an average of
46.5% of their starting weight during the study. Hamsters in the
group receiving SCV-07 at 10 .mu.g/kg on days -1 to 20 gained an
average of 51.3% of their starting weight during the study.
Hamsters in the group receiving SCV-07 at 100 .mu.g/kg on days -1
to 20 gained an average of 46.7% of their starting weight during
the study. Hamsters in the group receiving SCV-07 at 1 mg/kg from
day -1 to day 20 gained an average of 48.8% of their starting
weight during the study. The significance of these differences was
evaluated by calculating the area-under-the-curve (AUC) for the
weight gain of each animal, and then comparing the different
treatment groups using a One-Way ANOVA test. The results of this
analysis indicated that there were no significant differences
between the different treatment groups (P=0.663).
6.3 Mucositis (Tables 15 & 16)
[0137] The kinetics and severity of mucositis development among
control animals was consistent with that which was expected.
[0138] Mean daily mucositis scores for each group were evaluated.
In the saline treated control group, the mean peak mucositis score
was 3.1, which occurred on day 18. The group receiving SCV-07 at 10
.mu.g/kg from day -1 to day 20 had a peak mean mucositis score of
2.9, which occurred on day 16. The group receiving SCV-07 at 100
.mu.g/kg from day -1 to day 20 had a peak mean mucositis score of
2.8, which occurred on days 14 and 16. The group receiving SCV-07
at 1 mg/kg from day -1 to day 20 had a peak mean mucositis score of
3.2, which occurred on day 16. The significance of the differences
observed between the different treatment groups was evaluated by
calculating the number of days with a score of 3 or higher for each
group and comparing these numbers using a chi-squared
(.quadrature..sup.2) test. The results of this analysis are shown
in Table 15. The hamsters in the saline treated control group had a
score of 3 or higher on 50% of the animal days evaluated. In the
group receiving SCV-07 at 10 .mu.g/kg from day -1 to day 20, a
mucositis score of 3 or higher was observed on 34.2% of the animals
days evaluated, which was not statistically significantly different
from controls (P<0.001). In the group receiving SCV-07 at 100
.mu.g/kg on days -1 to 20, a mucositis score of 3 or higher was
observed on 29.2% of the animals days evaluated, which was
statistically significantly different from controls (P<0.001).
In the group receiving SCV-07 at 100 .mu.g/kg on days -1 to 20, a
mucositis score of 3 or higher was observed on 30.8% of the animals
days evaluated, which was not statistically significantly different
from controls (P<0.001). A further analysis of the mucositis
scores was performed using the Mann-Whitney rank sum analysis to
compare the scores for each group on each day. The results of this
analysis are shown in Table 16. In this analysis, 2 days of
significant reduction in the mucositis score are generally required
before it is regarded as meaningful. The group treated with SCV-07
at 10 .mu.g/kg on days -1 to 20, had significantly less mucositis
than the saline controls on days 22 (P=0.010), 24 (P=0.022), 26
(P=0.015), and 28 (P<0.001). The group treated with SCV-07 at
100 .mu.g/kg on days -1 to 20, had significantly less mucositis
than the saline controls on days 6 (P=0.015), 22 (P=0.003), 24
(P=0.005), 26 (P<0.001), and 28 (P<0.001). The group treated
with SCV-07 at 1 mg/kg on days -1 to 20, had significantly less
mucositis than the saline controls on days 6 (P=0.015), 20
(P=0.029), 22 (P=0.012), 24 (P=0.001), 26 (P=0.003), and 28
(P<0.001).
TABLE-US-00021 TABLE 15 SCI-04. Chi-square analysis of the total
number of days the animals in each group spent with a score of
three or more. This statistic is a measure of severity of
ulceration, a clinically important outcome. Days Days Total % Days
Chi Sq Group >=3 <3 Days >=3 v control P Value Vehicle,
qd, sc 120 120 240 50.0 -- -- Days -1 to 20 SCV-07 82 158 240 34.2
11.702 <0.001 10 ug/kg, qd, sc Days -1 to 20 SCV-07 70 170 240
29.2 20.9160 <0.001 100 ug/kg, qd, sc Days -1 to 20 SCV-07 74
166 240 30.8 17.5190 <0.001 1 mg/kg, qd, sc Days -1 to 20
TABLE-US-00022 TABLE 16 SCI-04. The significance of group
differences observed in daily mucositis scores was determined using
the Mann-Whitney rank sum test. This nonparametric statistic is
appropriate for the visual mucositis scoring scale. The p values
for each calculation are shown. Significant reductions in mucositis
scores relative to controls are shown underlined. Day Group
Comparison 6 8 10 12 14 16 18 20 22 24 26 28 Control vs 0.296 0.422
0.393 0.560 0.271 0.488 0.179 0.100 0.010 0.022 0.015 <0.001
SCV-7 10 ug/kg, qd, sc Days -1 to 20 Control vs 0.015 0.422 0.510
0.957 0.506 0.248 0.100 0.455 0.003 0.005 <0.001 <0.001 SCV-7
100 ug/kg, qd, sc Days -1 to 20 Control vs 0.015 0.282 0.146 0.924
0.506 0.634 0.112 0.029 0.012 0.001 0.003 <0.001 SCV-7 1 mg/kg,
qd, sc Days -1 to 20
7. Conclusions
[0139] 1. There was no evidence of any toxicity from SCV-07 in this
study based on the observations of mortality and weight gain. 2.
Animals treated with SCV-07 at 10 .mu.g/kg from day -1 to day 20
showed a statistically significant reduction in the number of days
with a mucositis score of 3 or higher (P<0.001), and a
significant reduction in mucositis scores on days 22 (P=0.010), 24
(P=0.022), 26 (P=0.015), and 28 (P<0.001). 3. Animals treated
with SCV-07 at 100 .mu.g/kg from day -1 to day 20 showed a
statistically significant reduction in the number of days with a
mucositis score of 3 or higher (P<0.001), and a significant
reduction in mucositis scores on days 6 (P=0.015), 22 (P=0.003), 24
(P=0.005), 26 (P<0.001), and 28 (P<0.001). 4. Animals treated
with SCV-07 at 1 mg/kg from day -1 to day 20 showed a statistically
significant reduction in the number of days with a mucositis score
of 3 or higher (P<0.001), and a significant reduction in
mucositis scores on days 6 (P=0.015), 20 (P=0.029), 22 (P=0.012),
24 (P=0.001), 26 (P=0.003), and 28 (P<0.001).
9. Appendices
TABLE-US-00023 [0140] APPENDIX 7 Animal Weights DAY Group Animal -1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 105 104 100 103 106 108 111
111 113 113 115 116 118 121 119 121 1 2 88 86 83 89 92 94 96 97 101
103 106 105 104 103 104 108 1 3 90 92 88 91 95 99 101 103 104 92
105 109 109 108 107 109 1 4 87 86 83 87 90 93 96 98 100 102 105 106
106 109 109 110 1 5 106 101 99 105 107 111 113 113 117 118 120 122
120 121 120 124 1 6 82 83 79 82 84 86 87 89 90 97 96 97 97 98 96 95
1 7 86 85 84 85 88 90 93 94 95 97 98 99 100 101 103 104 1 8 82 81
78 83 86 89 92 93 98 105 105 103 103 107 106 107 1 9 90 89 91 93 95
101 101 103 104 106 107 111 112 113 114 118 1 10 108 109 104 105
110 112 117 118 120 122 123 126 128 129 124 126 2 11 77 77 74 78 82
83 86 88 90 92 94 96 98 98 98 99 2 12 101 102 101 104 111 113 112
114 117 121 122 125 125 128 128 130 2 13 85 84 83 89 93 95 98 101
103 106 106 112 111 111 112 115 2 14 82 80 80 86 88 90 93 96 98 101
102 105 105 107 108 109 2 15 76 77 76 77 79 82 83 84 86 88 91 90 92
91 90 91 2 16 94 94 91 94 98 101 103 106 108 109 108 109 109 109
108 111 2 17 74 72 70 77 79 76 81 79 80 83 83 85 87 88 88 90 2 18
80 78 75 78 81 85 85 89 89 92 92 94 95 94 95 96 2 19 91 90 87 90 91
93 94 94 97 98 98 100 100 97 98 99 2 20 92 92 90 95 96 99 100 102
105 106 108 110 111 113 114 114 3 21 87 84 87 90 94 98 99 98 100
103 106 111 109 111 111 110 3 22 81 80 75 77 81 84 86 88 87 90 91
95 95 98 96 97 3 23 85 86 83 86 88 91 93 96 95 98 96 101 100 102
102 103 3 24 104 102 99 102 106 109 113 112 116 118 118 123 123 126
127 130 3 25 78 76 73 76 82 83 84 84 87 90 89 95 94 92 92 95 3 26
92 91 88 90 91 92 95 95 96 98 98 100 100 101 100 100 3 27 103 103
102 100 108 110 115 118 119 120 120 124 124 127 125 128 3 28 96 96
91 90 101 114 105 106 107 108 111 113 114 117 116 116 3 29 83 82 84
87 91 91 94 94 97 98 97 103 101 102 102 101 3 30 77 78 74 76 78 79
81 83 86 88 88 92 94 93 94 94 4 31 97 96 94 98 103 106 108 110 111
112 112 113 111 113 113 114 4 32 96 96 93 95 99 103 104 106 109 110
111 113 113 113 114 113 4 33 93 91 89 90 94 100 101 103 107 110 110
114 115 114 113 113 4 34 87 85 85 89 92 96 98 103 104 120 107 109
112 113 113 115 4 35 100 96 97 101 106 108 111 115 118 105 121 124
122 126 124 124 4 36 88 88 85 87 93 96 99 103 105 108 109 113 114
118 120 121 4 37 104 102 104 108 114 115 120 119 123 125 127 128
127 127 122 125 4 38 97 96 93 93 98 103 105 107 110 113 113 116 116
116 115 114 4 39 88 88 83 85 93 93 95 95 98 100 102 104 102 104 104
103 4 40 99 97 99 101 107 107 112 113 115 117 118 120 123 125 125
127 DAY Group Animal 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1 1
119 121 124 125 126 127 130 133 133 137 137 138 139 140 1 2 112 110
117 116 120 121 124 125 123 131 134 136 135 139 1 3 112 117 116 117
118 120 126 127 129 131 132 135 134 138 1 4 110 109 113 115 115 115
121 120 121 126 126 129 129 133 1 5 123 125 129 129 129 130 133 136
135 141 141 143 144 144 1 6 95 96 97 99 99 100 102 107 104 106 107
108 109 111 1 7 103 104 107 107 108 108 111 113 113 115 118 120 120
122 1 8 108 109 113 115 115 115 119 122 123 125 127 128 129 132 1 9
118 117 122 123 123 126 128 130 131 134 135 136 138 138 1 10 126
128 131 134 134 135 137 139 141 144 147 148 148 151 2 11 99 100 103
103 105 104 108 110 110 114 115 117 117 119 2 12 132 132 136 137
138 137 141 144 143 148 150 151 152 156 2 13 117 119 123 125 125
127 130 133 133 139 139 140 145 146 2 14 109 109 112 113 113 115
117 120 121 123 124 126 127 130 2 15 93 92 96 98 100 100 103 104
104 105 108 109 110 113 2 16 112 112 117 117 118 118 121 122 123
126 126 129 128 132 2 17 90 90 96 97 99 101 105 104 106 109 113 114
114 117 2 18 97 96 103 102 101 109 105 108 108 111 112 116 114 116
2 19 98 99 100 104 103 104 106 107 108 111 113 115 113 115 2 20 115
117 121 122 123 123 127 131 129 134 136 137 138 143 3 21 109 110
112 114 115 118 117 123 123 128 127 130 130 133 3 22 97 98 100 102
103 104 107 109 108 113 112 115 112 117 3 23 103 103 106 107 110
110 113 114 113 117 117 123 119 123 3 24 131 132 134 134 140 139
142 146 146 146 148 149 149 154 3 25 95 97 99 101 101 105 106 108
106 113 115 118 116 120 3 26 110 101 103 104 105 106 106 109 109
112 112 113 113 116 3 27 128 128 129 132 134 136 138 139 138 141
143 146 146 149 3 28 116 117 119 120 123 123 126 128 128 131 133
132 133 137 3 29 103 105 106 108 112 112 117 118 117 123 123 125
126 130 3 30 95 97 100 101 102 102 104 107 107 111 114 116 115 118
4 31 112 113 114 116 117 120 121 123 124 130 130 133 133 136 4 32
123 116 118 119 119 122 123 125 124 129 130 132 133 136 4 33 112
114 117 119 122 122 126 129 128 133 137 138 138 141 4 34 116 116
120 120 123 122 125 126 127 129 131 131 131 133 4 35 114 123 127
130 130 132 137 141 139 142 147 147 145 150 4 36 118 119 120 123
123 125 128 130 129 134 136 139 137 142 4 37 125 128 131 132 134
135 139 143 143 145 148 150 150 153 4 38 115 115 119 119 121 122
123 125 126 128 130 133 132 135 4 39 105 107 110 112 114 114 115
117 116 119 123 126 125 130 4 40 125 128 131 134 135 133 138 142
144 148 149 152 151 155
TABLE-US-00024 APPENDIX 8 Mucositis Scores Ani- DAY Group mal 6 8
10 12 14 16 18 20 22 24 26 28 1 1 2 0 1 2 3 3 3 3 3 3 3 3 1 1 1 0 1
2 3 3 3 3 3 3 3 3 1 2 1 1 2 3 3 3 3 3 3 2 2 2 1 2 0 0 2 3 3 3 4 3 3
2 2 2 1 3 2 1 2 3 4 4 3 3 3 3 3 3 1 3 1 1 2 3 3 4 3 3 3 3 3 3 1 4 0
0 1 2 2 3 3 3 3 3 3 3 1 4 0 0 1 2 2 3 3 3 3 3 3 3 1 5 2 1 2 3 3 3 3
3 3 3 3 3 1 5 1 1 2 2 3 3 3 3 3 3 3 3 1 6 1 0 1 2 3 3 3 3 3 2 2 2 1
6 0 0 1 2 3 3 3 3 3 2 2 2 1 7 0 0 1 1 2 2 2 2 2 2 1 1 1 7 0 0 0 1 2
2 2 2 2 2 1 1 1 8 1 0 1 2 3 3 3 3 3 2 2 2 1 8 0 0 1 2 3 3 4 3 3 2 2
2 1 9 0 0 1 3 3 3 3 3 3 2 2 1 1 9 0 0 1 3 3 3 3 3 2 2 2 1 1 10 0 0
1 2 4 3 4 3 3 3 2 2 1 10 0 0 0 2 4 4 4 3 3 3 2 2 2 11 0 0 1 2 2 3 2
3 2 2 1 0 2 11 0 0 1 2 2 3 2 3 2 1 1 0 2 12 1 0 2 2 2 2 2 2 1 1 1 0
2 12 0 0 1 2 2 2 2 2 1 1 1 0 2 13 1 0 1 3 3 3 3 2 2 2 1 0 2 13 0 0
1 3 3 3 3 1 2 2 1 0 2 14 1 0 1 2 2 3 3 3 3 3 3 2 2 14 0 0 1 2 2 3 3
3 3 3 3 2 2 15 1 0 1 2 3 3 3 3 3 2 2 1 2 15 0 0 1 2 3 3 3 3 3 2 1 1
2 16 1 1 2 3 3 2 2 2 1 1 1 0 2 16 0 0 2 3 3 2 2 2 1 1 1 1 2 17 1 0
2 3 3 4 3 3 2 2 2 1 2 17 0 0 2 3 3 4 3 3 2 2 2 1 2 18 0 0 2 2 3 3 3
2 2 3 1 1 2 18 0 0 1 2 3 3 3 2 2 3 1 1 2 19 0 0 1 3 3 3 4 3 3 2 3 3
2 19 0 0 1 3 3 3 4 3 3 2 3 3 2 20 0 1 2 2 3 3 3 3 3 2 2 2 2 20 0 0
2 2 3 3 3 3 3 2 2 1
* * * * *