Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITION AND METHOD FOR
TREATING HEMATOLOGIC CANCERS
Description
Field of the Invention
This invention relates to a therapeutic
regimen for treating blood (hematologic) cancers such
as leukemia, lymphoma, and multiple myeloma, and
particularly effecting such treatments in children.
BACKGROUND ART
An adult human has about 7000 white blood
cells per microliter ( L) of blood. Of those white
cells, about 65 percent are granulocytes (about
4500/ L) , about 30 percent are monocytes (about
2100/ pi) and about five percent are lymphocytes (about
350/ EiL) . Geyton, Textbook of Medical Physiology,
Seventh ed., W. B. Saunders Co., Philadelphia (1986).
The above cell numbers are, of course, generalized
average values, and granulocyte counts for normal
patients; i.e., patients free of disease, typically
have granulocyte counts of about 2000 to about 7000
cells/ L.
Chronic myelogenous leukemia (CML), also
known as chronic granulocytic leukemia (CGL), is a
neoplastic disorder of the hematopoietic stem cell. In
its early phases it is characterized by leukocytosis,
- the presence of increased numbers of immature
granulocytes in the peripheral blood, splenomegaly and
anemia. These immature granulocytes include basophils,
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eosinophils, and neutrophils. The immature
granulocytes also accumulate in the bone marrow,
spleen, liver, and occasionally in other tissues.
Patients presenting with this disease
characteristically have more than 75,000 white blood
cells per microliter ( L), and the count can exceed
500,000/ L.
CML accounts for about 20 percent of all
leukemias in the United States. About 15 new cases per
million people are reported each year, leading to about
3,000 to 4,000 new cases per year. The disease is rare
in humans below age 45, rises rapidly to age 65, and
remains high thereafter. The median life span of
patients with chronic myelogenous leukemia from the
time of diagnosis is approximately four years.
About 60 to 80 percent of patients with CML
develop a blast crisis. Blast crisis represents a
manifestation of acute leukemia. The presence of
certain markers on the blast cells sometimes suggests a
lymphold origin of these cells during the blast crisis.
Chemotherapeutic agents used for the
treatment of the blast crisis are the same as those
used for the treatment of other acute leukemias. For
example, cytarabine and daunorubicin, used for the
treatment of acute myelocytic leukemia, are used to
treat CML blast crisis. Prednisone and vincristine, a
therapeutic regime used in the treatment of acute
lymphocytic leukemias, is also used to treat CML blast
crisis. Nevertheless, these drug therapies of the
blast crisis stage of CML are even less successful than
are the treatments of other acute leukemias.
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Cancer in children is rare with an incidence
of 140-155 per million per year (age million per year
(age <15 years). This translates to -1 in 7,000
children is diagnosed with cancer each year. Despite
the rarity of cancer, malignant neoplasm is the most
common cause of death after accidents in children aged
to 14 years, accounting for 23% of mortality.
Survival from childhood cancers, many of which were
fatal in the pre-chemotherapy era, has increased
dramatically from 20-30% in the 1960s to 62% in the
1970s, and more recently to 83%. Saletta et al.,
Transl Pediatr 3(2):156-182 (2014).
Leukemias are the most common childhood
cancers, accounting for about 30% of all pediatric
(ages 1-14) cancer diagnoses. Acute lymphoblastic
leukemia (ALL) accounts for about 25% of ohildrens'
cancers, and acute myeloid leukemia (AML) accounts for
the remaining about 5%. Non-hodgkin lymphoma (NHL) and
Hodgkin lymphoma account for about 6 and about 4 % of
childhood cancers, respectively. (Ibid.)
Current treatments for ALL include pegylated
asparginase, liposomal daunorubicin, liposomal
annamycin, sphingosomal vincristine and liposomal
cytarabine. For AML, current treatments include the
use of all-trans-retinoic acid (ATRA), arsenic
trioxide, anthracycline combined with ATRA, and
idarubicin with high-dose cytarabine. Sorafenib
(multikinase inhibitor) in combination with clofarabine
and cytarabine has found success in a phase I study [
Inaba et al., J din Oncol 29:3293-3300 (2011)], and a
calicheamicin-conjugated CD33 antibody, gemtuzumab
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ozogamicin, known commercially as MylotargO, has shown
promise [Zwaan et al., Br J Baemato1 148:768-776
(2010)].
Unlike adults, in whom non-Hodgkin's lymphoma
(NHL) is generally low/intermediate grade, pediatric
NHL is frequently high grade. NHL can be classified
according to phenotype (B-cell vs. T-cell) and
differentiation. It falls into three categories: (I)
mature B-cell NHL including Burkitt/Burkitt-like
lymphoma and diffuse large B-cell lymphoma (DLBCL);
(II) lymphoblastic lymphoma (LL) (mostly precursor
T-cell); and (III) anaplastic large cell lymphoma
(ALCL) (mature T-cell or null-cell). Burkitt lymphoma
(BL) is most common, accounting for one-third of
pediatric NHL [Saletta et al., Transl Pediatr 3(2):156-
182 (2014)1.
Rituximab (CD 20 antibody) alone and in
conjunction with chemotherapy has been undergoing
trials in patients with BL and DLBCL [windebank et al.,
Pediatr Blood Cancer 53:392-396 (2009); and Gross et al., Am
J Transplant 12:3069-3075 (2012)]. Brentuximab vedotin a
chimer of CD30 antibody and monomethyl auristatin E has
shown positive responses and remissions in adult phase
II trials [Pro et al., J din Oncol 30:2190-2196
(2012)], whereas erizotinib and ALK (anaplastic
lymphoma kinase) inhibitor elicited positive responses
in 8/9 ALCL patients in a phase I study [Mosse' et al.,
Lancet Oncol 14:472-480 (2013)1.
Hodgkin's lymphoma (HL) is the most common
cancer in the 15 to 19 years age group and is four to
five times more frequent than in the <15 years age
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group. HL is typically categorized into classical and
nodular lymphocyte predominant HL.
HL was fatal until the 1960s when the MOPP
(nitrogen mustard, vincristine, procarbazine and
prednisone-containing) chemotherapy regimen was
introduced. The cure rate of HL in children has been
>90% in the last two decades and is one of the most
curable childhood cancers. Unfortunately, survivors of
childhood HL are at significant risk of long-term
treatment-related morbidity and mortality.
Earlier treatments typically included use of
radiation along with chemotherapy, which increased the
subsequent finding increased breast cancers in
previously treated women. More modern approaches have
limited use of radiation in patients in complete
response after two cycles of chemotherapy referred to
as rapid early responder (PER), omission of involved-
field radiation therapy (IFIRT) in low risk RER, and
gender-based modification of treatment to utilize less
gonadal toxic alkylating therapy in males ad avoiding
IFRT in females.
Although the survival rate for pediatric
leukemia has greatly improved, relapse is a major cause
of treatment failure. Approximately 15-20% of
pediatric ALL patients and 30-40% of AML patients
relapse, with relapsed ALL identified as the fourth
most common malignancy in children.
Treatment of relapsed pediatric leukemia
includes intensification of chemotherapeutic regimens
and use of bone marrow transplantation (EMT). However,
increasing the intensity of combination chemotherapies
and introduction of second-line drugs is often
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accompanied by cumulative toxicity with marginal
incremental benefits.
A key component for understanding immune
system interactions against pediatric cancers is the
availability of an applicable animal model. Current
xenograft models are limited because they are
established in severe combined immunodeficient (SCID)
mice and so do not provide information on the
contribution of the immune system. Other approaches
such as human hematopoietic stem cell reconstitution in
immunocompetent animals are cumbersome, expensive and
often introduce complex biological variables into the
system.
Recently, a novel xenograft tumor model was
developed in immunocompetent mice by tolerizing mice
fetuses to human tumor cells [Basel et al., Cancer
Lett. 412:256-263 (2018)]. This model is advantageous
because it can be used to better describe the complex
interaction between cancerous cells and the immune
system through a xenograft technique.
One useful anti-cancer agent group for adult
cancerous tumors are the halogenated xanthenes, or the
pharmaceutically acceptable salts thereof. See, US
Patents No. 6,331,286, No. 7,390,668, No. 7,648,695,
No. 9,107,887, No. 9,808,524, No. 9,839,688, and No.
10,130,658. Of those halogenated xanthenes, Rose
Bengal disodium, (4,5,6,7-tetrachloro-2',4',5',7'-
tetraiodofluorescein disodium; RB) has been found to be
particularly effective and easily utilized.
PV-10 is a sterile 10% solution of RB in 0.9%
saline that has been used clinically to measure liver
function in infants [Yvart et al., Eur J Nucl _Pied
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6:355-359 (1981)]. Previous studies have shown that
PV-10 accumulates in cancer cell lysosomes [Wachter, et
al., Proceedings or SPIE, Multiphoton Microscopy in the
Biomedical Sciences II, Periasamy, A. and So, P.T.C.
(eds), Bellingham, Washington: 4620: 143-147 (2002)]
and induces cell death in a range of adult cancers [Qin
et al., Cell Death Dis 8:e2584 (2017); Toomey et al.,
FLoS ONE 8(7):e68561 (2013); Koevary et al., Int J
Physiol Pathophysiol Pharmaco1 4(2):99-107 (2012);
Thompson et al., Melanoma Res 18(6):405-411 (2008); and
Zamani et al., J Immunotoxic 11(4):367-375 (2014)].
PV-10 has been used in a number of clinical
trials, both as a single anti-cancer agent and in
conjunction with both small molecule and monoclonal
antibody anti-cancer agents. Several of those trials
are discussed below. Phase I and phase IT clinical
studies using PV-10 alone as the cytotoxic agent
illustratively reported "adverse events were
predominantly mild to moderate and locoregional to the
treatment site, with no treatment-associated grade 4 or
adverse events" [Thompson et al., Ann Surg Oncol
22(7):2135-2142 (2015)11 and "Treatment-Emergent
Adverse Events (TEAEs) were consistent with established
patterns for each drug, principally Grade 1-2 injection
site reactions attributed to PV-10 and Grade 1-3
immune-mediated reactions attributed to pembrolizumab,
with no significant overlap or unexpected toxicities:
. " [Agarwala et al., J din Oncol 37(15) suppl 9559-
9559 (May 26, 2019)1. It thus appears as though RB is
toxic to cancerous cells, but non-toxic to non-
cancerous cells.
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Because of the often-times very different
behavior of adult tumors from pediatric tumors, it was
not known whether RB and similar halogenated xanthenes
would be effective when used against pediatric
cancerous cells, and particularly cancerous hematologic
cells. Preliminary in vitro and xenograft studies
against neuroblastoma cell lines in cell cultures to
which RB was added alone or in conjunction with known
anticancer agents, and by intralesional injection in
mice, respectively, were reported by one of the present
inventors and co-workers to exhibit killing of the
cancerous cells. Swift et al., Oncotargets Ther,
12:1293-1307 (February 2019).
In addition, intralesional administration of
a halogenated xanthene into a tumor provides the active
cytotoxic agent directly to the tumor at its highest
concentration. In a presently contemplated treatment
technique discussed below, administration is often
distant from the target cancerous hematiologic cells,
thereby possibly diminishing the effectiveness of the
cancerocidal halogenated xanthene agent.
However, in a phase II clinical trial for
patients with refractory metastatic melanoma,
intralesional (IL) injection of PV-10 induced tumor
regression with an overall response rate of 51%
[Thompson et al., Ann Surg Oncol 22(7):2135-2142
(2015)]. PV-10 also demonstrated efficacy in
combination with radiotherapy in a phase II clinical
trial for patients with in-transit or metastatic
melanoma, with an overall response rate of 86.6% [Foote
et al., J Surg Oncol 115(7): 891-897 (2017)].
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In addition to inducing direct cancer cell
death, PV-10 has also been shown to induce a tumor-
specific immune response in both mouse studies [Qin et
al., Cell Death This 8:e2584 (2017); Toomey et al., PLoS
ONE 8(7):e68561 (2013); and Liu et al., Oncotarget
7(25):37893-37905 (2016)] and human clinical trials
fLippey et al., J Surg Oncol 114(3):380-384 (2016);
Ross, J Surg Oncol 109(4):314-319 (2104); Liu et al.,
PLoS ONE 13(4):e0196033 (2018); andBasel et al., Cancer
Lett 412:256-263 (2018)]. In murine models of melanoma,
treatment with PV-10 induced necrosis of melanoma cells
and a localized increase in mononuclear tumor-
infiltrating lymphocytes [Lippey et al., J Surg Oncol
114(3):380-384 (2016)].
It has been suggested that PV-10-induced
immunogenic cell death, releasing tumor antigens to
nearby antigen-presenting cells (APCs), facilitated the
activating of anti-tumor T and B cells. In a syngeneic
murine colon cancer model, injection of cancer cells
treated in vitro with PV-10 into mice with the same
tumor resulted in slower tumor growth [Oin et al., Cell
Death Dis 8:e2534 (2017)]. Furthermore, in syngeneic
murine melanoma models, combination treatment with
intralesional PV-10 and anti-PD1 antibody delayed tumor
growth and enhanced T cell activation [Liu et al., PLoS
ONE 13(4):e0196033 (2108)].
The disclosure below describes the
contemplated invention and provides results of studies
using halogenated xanthenes such as PV-10 in the
treatment of pediatric leukemias.
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BRIEF SUMMARY OF THE INVENTION
The present invention contemplates a method
of treating a mammalian subject having hematologic,
non-tumorous cancer cell. Illustrative hematologic,
non-tumorous cancers include leukemia, lymphoma and
myeloma. The method comprises the steps of: (A)
administering to such a mammalian subject a
therapeutically effective amount of a halogenated
xanthene, a pharmaceutically acceptable salt or a 01-04
alkyl ester thereof as a first cancer cytotoxic agent
dissolved or dispersed in a pharmaceutically acceptable
aqueous medium. The mammalian subject is maintained
for a period of time sufficient to induce death of
hematologic, non-tumorous cancer cells. A contemplated
administration is typically repeated.
A contemplated treatment method can also be
carried out in conjunction with administration to that
mammalian subject of a second therapeutically effective
amount of a second, differently-acting cancer cytotoxic
agent dissolved or dispersed in a pharmaceutically
acceptable medium_ The second cancer cytotoxic agent
can be a small molecule or an intact antibody or
paratope-containing antibody portion. The first and
the second cancer cytotoxic agents can be administered
together in the same or different media, or at
different times. The second cancer cytotoxic agent can
be administered in a solid tablet, capsule, pill or the
like or in a liquid medium.
In one aspect, use of a small molecule cancer
cytotoxic agent having a molecular weight of about 200
to about 1000 pa is contemplated. Compounds that
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synergize with a halogenated xanthene such as
doxorubicin, etoposide and vincristine are preferred.
Intact antibodies or paratope-containing antibody
portions are a second group of cancer cytotoxic agents.
Preferred among these agents are those referred to as
immune check point inhibitors. [See, for example,
Darvin et al., Exp Hal Med, 50:165 (2018).]
The present invention also contemplates use
of a therapeutically effective amount of a halogenated
xanthene, a pharmaceutically acceptable salt or a Ci-C4
alkyl ester thereof as a first cancer cytotoxic agent
dissolved or dispersed in a pharmaceutically acceptable
aqueous medium for treatment of a mammalian subject
having hematologic, non-tumorous cancer cells, wherein
the halogenated xanthene is maintained in the mammalian
subject for a period of time sufficient to induce death
of hematologic, non-tumorous cancer cells. In a
further embodiment, the first cancer cytotoxic agent
halogenated xanthene, pharmaceutically acceptable salt
or C1-04 alkyl ester thereof is rose bengal, a
pharmaceutically acceptable salt or C1-C4 alkyl ester
thereof. In a still further embodiment, the rose
bengal is rose bengal disodium salt. Further, the
hematologic, non-tumorous cancer cells are leukemia,
lymphoma or myeloma. Further, the hematologic, non-
tumorous cancer cells, leukemia, lymphoma or myeloma is
selected from the group consisting of acute B-cell or
T-cell lymphoblastic leukemia, acute myeloid leukemia,
non-Hodgkin's lymphoma, and Hodgkin's lymphoma.
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DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In one aspect, the present invention
contemplates a pharmaceutical composition for use in
the treatment (killing) of hematologic, non-tumorous
cancer cells, e.g., leukemia, lymphoma and myeioma. A
contemplated pharmaceutical composition comprises a 0.1
% to about 20 % (w/v) aqueous medium (as a liquid) of a
first cancer cytotoxic agent that is a halogenated
xanthene, a physiologically acceptable salt of the
halogenated xanthene, or a C1-C4 alkyl ester thereof.
A particularly preferred halogenated xanthene salt is
rose bengal (4,5,6,7-tetrachloro-2',4',5',7t-
tetraiodofluorescein) disodium salt, as is present in
PV-10. The composition is administered to provide a
therapeutically effective amount of a first cancer
cytotoxic agent to a mammal such as a human having a
hematologic, non-tumorous cancerous disease such as
leukemia, lymphoma and myeloma, or more specifically,
acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), non-Hodgkin's lymphoma (NHL), or
Hodgkin's lymphoma (HL).
The mammalian subject is maintained for a
time sufficient to kill hematologic, non-tumorous
cancerous cells. The fact and relative amount of
cancer killing can be determined by usual means for
assaying the status of hematologic, non-tumorous
cancers.
The mammalian subject is typically thereafter
treated again, usually multiple times. Both the
duration of maintenance and the choice to conduct
further administrations can depend upon the species of
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mammal, individual mammalian subjects, the severity of
disease, type of disease, age and health of the
subject, and the like. These factors are commonly
dealt with by physicians skilled in the art of treating
hematologic, non-tumorous cancers.
In addition, whereas it is typically desired
to rid the body of detectable cancerous cells, that
cannot always be done. Sometimes it is sufficient to
kill enough cancerous cells to control the disease in
stasis, or to reduce the cancerous load of cells so
that other therapies can be carried out.
The data provided hereinafter illustrates
that the I050 value for use of RB against several
leukemia cell lines in vitro is about 50 to about 100
M. Given that the molecular weight of RB is 1018
g/mole, the above 1050 value calculates to about 50 to
about 100 mg of RB/liter. It is preferred to achieve
that concentration for contacting cancerous cells
during an in vivo treatment.
The classic intravenous (IV) diagnostic use
of RB was conducted giving 100 mg RB as a single IV
dose. In clinical studies of PV-10, RB has been
tolerated at 1500 mg delivered IV. The standard adult
blood volume is approximately 5 L. Thus, to achieve
100 mg/L in the blood, an adult patient would need to
receive approximately 500 mg of RB IV to achieve the
1050 value in the bloodstream. Due to the rapid
clearance of RB from circulation (t1/2 is about 30
minutes), an IV administration would require continuous
infusion to maintain peak levels of RB in circulation
(i.e., for up to several hours or more).
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Administration at the IC50 value level would
not be toxic to all circulating hematologic, non-
tumorous cancerous cells; i.e., only approximately half
of cells would be affected at the 1050 value. It can
therefore be preferred to administer RB at a multiple
of the IC50 value, up to approximately 1500 mg (i.e.,
300 M).
Alternatively, it can be sufficient to kill
only a fraction of the cancerous cells to initiate a
functional immune response against remaining tumor
burden. The latter case can be preferable to avoid
toxic reaction (i.e., so-called "tumor lysis syndrome"
due to presence of an abundance of rapidly killed
cancer cells. In this situation, the cancer cell
debris caused by the cytotoxicity to cancer cells of a
halogenated xanthene induces an immune reaction that in
turn kills further cancerous hematologic, non-tumorous
cells.
The similarly useful halogenated xanthene
compounds listed below and their pharmaceutically
acceptable salts can have molecular weights that differ
from each other by about a factor of three (See, Table
3, US Patent No. 7,390,688 at columns 15-16). It is
preferred that an exact amount of other than RB
halogenated xanthene to be used is calculated based on
published molecular weights for each such compound and
that of RB.
A contemplated halogenated xanthene includes
rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodo-
fluorescein) that is particularly preferred, erythrosin
B, phloxine B, 4,5,6,7-tetrabromo-2',4',5',7'-tetra-
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iodofluorescein, 21,4,5,6,7-pentachloro-4',5'77'-
triiodofluorescein, 4,4',5,6,7-pentachloro-2',5'17'-
triiodofluorescein, 21,4,5,6,7,7'-hexachloro-4',5'-
diiodofluorescein, 4,4',5,5',6,7-hexachloro-2'17'-
diiodofluorescein, 2',4,5,5',6,7-hexachloro-4',7'-
diiodofluorescein, 4,5,6,7-tetrachloro-2',4',5'-
triiodofluorescein, 4,5,6,7-tetrachloro-2'14',7'-
triiodofluorescein, 4,5,6,7-tetrabromo-27,47,57-
triiodofluorescein, and 4,5,6,7-tetrabromo-2'14',7'-
triiodofluorescein. The reader is directed to Berge,
J. Pharm. Sci. 1977 68(1):1-19 for lists of commonly
used pharmaceutically acceptable acids and bases that
form pharmaceutically acceptable salts with
pharmaceutical compounds.
A C1-C4 alkyl ester of one of the above
halogenated xanthene compounds can also be used, with
the C2; i.e., ethyl ester, being preferred. Thus, in
vitro studies using each of RB, ethyl-Red 3
(erythrosine ethyl ester; 2',4',5',7'-tetraiodo-
fluorescein ethyl ester), 4,5,6,7-tetrabromo-
2',4',5',7'-tetraiodofluorescein and ethyl-Pholoxine B
(4,5,6,7-tetrachloro-2',4',5',7'-tetrabromofluorescein
ethyl ester) exhibited similar anti-tumor activities
against CCL-142 renal adenocarcinoma.
A preferred form of rose bengal is rose
bengal disodium that has the structural formula below:
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CI
a
a
a
COONa
Na0 0!O.
Further details of the medicinal use a pharmaceutical
composition containing an above-noted halogenated
xanthine are described in U.S. Patents No. 5,998,597,
No. 6,331,286, No. 6,493,570, No. 7,390,688, No.
7,648,695, No. 8,974,363, No. 9,107,887, No. 9,808,524,
No. 9,839,688, No. 10,130,658 and No. 10,471,144, whose
disclosures are incorporated by reference herein in
their entireties.
A contemplated halogenated xanthene or its
pharmaceutically acceptable salt is typically used
dissolved or dispersed in an aqueous pharmaceutical
composition. The halogenated xanthene is typically
present at 0.1 to about 20 % (w/v) in an aqueous 0.9 %
saline pharmaceutical composition.
Because a contemplated pharmaceutical
composition is typically intended for parenteral
administration as by intravenous methods, such a
composition should contain an electrolyte, and
preferably have approximately physiological osmolality
and pH value. A preferred concentration of singly
charged electrolyte ions in a pharmaceutically
acceptable aqueous medium is about 0.5 to about 1.5%
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(w/v), more preferably at about 0.8 to about 1.2%
(w/v), and most preferably at a concentration of about
0.9% (w/v). The about 0.9% (w/v) concentration is
particularly preferred because it corresponds to an
approximately isotonic aqueous solution. In a further
preferred embodiment, the electrolyte in a
chemoablative pharmaceutical composition is sodium
chloride.
Electrolytes at such levels increase the
osmolality of a pharmaceutically acceptable aqueous
medium. Thus, as an alternative to specifying a range
of electrolyte concentrations, osmolality can be used
to characterize, in part, the electrolyte level of the
composition. It is preferred that the osmolality of a
composition be greater than about 100 mOsm/kg, more
preferably that the osmolality of the composition be
greater than about 250 mOsm/kg, and most preferably
that it be about 300 to about 500 mOsm/kg.
It is preferred that the pH value of a
pharmaceutically acceptable aqueous medium be about 4
to about 9, to yield maximum solubility of the
halogenated xanthene in an aqueous vehicle and assure
compatibility with biological tissue. A particularly
preferred pH value is about 5 to about 8, and more
preferably between about 6 to about 7.5. At these pH
values, the halogenated xanthenes typically remain in
dibasic form, rather than the water-insoluble lactone
that forms at low pH values.
The pH value of a pharmaceutically acceptable
aqueous medium can be regulated or adjusted by any
suitable means known to those of skill in the art. The
composition can be buffered or the pH value adjusted by
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addition of acid or base or the like. As the
halogenated xanthenes, or physiologically acceptable
salts thereof, are weak acids, depending upon
halogenated xanthene concentration and/or electrolyte
concentration, the pH value of the composition may not
require the use of a buffer and/or pH modifying
reagent. It is especially preferred, however, that the
composition not contain any buffer, permitting it to
conform to the biological environment once
administered.
In the present invention, the specific amount
of halogenated xanthene in a pharmaceutical composition
is not believed to be as important as was the case
where the composition was injected intralesionally to a
tumor because the object here is to ultimately provide
a cytotoxic concentration of halogenated xanthene to
the environment of the cancerous cells and in which
those cancerous cells can be contacted with the
halogenated xanthene. The data provided hereinafter
indicate that an IC50 concentration of disodium rose
bengal is about 50 to about 100 LIM for in vitro cultured
leukemia cells.
The above results using in vitro cultured
leukemia cells surprisingly provided data similar to
those obtained in an in vitro cytotoxicity study of
cultured SK-N-AS, SK-N-BE(2), IMR5, LAN1, SHEP, and SK-
N-SH neuroblastoma cells, SK-N-MC neuroepithelioma
cells, and normal primary, BJ, and WI38 fibroblasts
reported by Swift et al., OncoTargets and Therapy 12:1-
15 (2019). Those authors reported half maximal
inhibitory concentration (IC50) values for PV-10-
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treated cells at 96 hours post treatment of 65-85 M for
the neuroblastoma lines assayed and 49 RN for the
neuroepithelioma line SK-N-MC. Those authors also
examined toxicity toward human epithelial cells from
three tissue sources and reported 1050 values of 93-143
M.
Presuming an IC50 value for leukemia cells of
about 50 to about 100 M, a dose to kill about one-half
of the leukemia cells in an adult would calculate to be
about 50 to about 100 mg of RB/L, based on a molecular
weight of 1018 9/mole for disodium RB. The classic IV
diagnostic use of RB was conducted giving 100 mg RB as
a single IV dose. The standard adult blood volume is
approximately 5 L. Thus, to achieve 100 mg/IL in the
blood, an adult patient would need to receive
approximately 500 mg of RE IV to achieve the IC50 value
in the bloodstream. Intravenous (IV) dosing is a
preferred method of administering a halogenated
xanthene-containing composition to a mammalian subject
in need.
In clinical studies of PV-10, RB has been
tolerated at 1500 mg delivered IV. Due to the rapid
clearance of RB from circulation (t1/2 about 30
minutes) an IV administration would require continuous
infusion to maintain peak levels of RB in circulation
(i.e., for up to several hours or more) during a single
administration.
Administration of sufficient RB to achieve a
circulating RB concentration at the IC50 level would
not be toxic to all circulating leukemic cells (i.e.,
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only approximately half of the leukemic cells would be
affected at the IC50). In some embodiments it can be
preferred to administer RB in an amount that is a
multiple of the IC50 level, up to approximately 1500 mg
(i.e., 300 mM). Alternatively, however, it can be
sufficient to kill only a fraction of tumor cells as a
result of an individual administration.
The latter case can be preferable for
avoiding a toxic reaction (i.e., tumor lysis syndrome)
that can result from rapidly killed tumor cell burden.
Thus, Howard et al., N Engl L7 Med 364(19):1844-1854
(May 12, 2011) report that tumor lysis syndrome is the
most common disease-related emergency encountered by
physicians treating hematologic cancers.
A mammal having leukemia, lymphoma or myeloma
in need of treatment (a mammalian subject) and to which
a pharmaceutical composition containing a halogenated
xanthene or its pharmaceutically acceptable salt can be
administered can be a primate such as a human, an ape
such as a chimpanzee or gorilla, a monkey such as a
cynomolgus monkey or a macaque, a laboratory animal
such as a rat, mouse or rabbit, a companion animal such
as a dog, cat, horse, or a food animal such as a cow or
steer, sheep, lamb, pig, goat, llama or the like.
As discussed in greater detail below, it can
also be advantageous to kill only a portion of the
leukemic cells during a single treatment to initiate a
functional immune response against remaining cancer
cell burden. A RB-initiated functional immune system
response is believed to occur due at least in part from
the action of RB-caused necrotic cell debris
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circulating in the body induces an immune response that
can prolong the effects of an initial administration of
a halogenated xanthene such as RB.
An induced immune response can take a longer
=
time to develop than the more immediate killing of the
contacted cancerous cells. That delay in effect can
occur because of the time needed for induction the
appropriate B and T cell populations to attack and kill
the leukemic cells as well as to induce long lasting
memory T cells whose continued circulation can protect
the patient from relapse.
In another aspect, an above pharmaceutical
composition is used in conjunction with a second,
differently-acting cytotoxic anticancer agent; i.e., a
cytotoxic anticancer agent whose mechanism of action is
different from that of the first cytotoxic agent, the
halogenaed xanthene. As noted previously, the
halogenated xanthenes localize in cancer cell
lysosomes, increase the percentage of cells in Cl phase
of the cell cycle and induces cell death by apoptosis
[Swift et al., Oncotargets Ther, 12:1293-1307 (February
2019)].
A first type of second cytotoxic agent is a
so-called 'small molecule". Such small molecules can
be viewed as semi-specific cellular poisons in that
they are generally more specific at killing cancer
cells than non-cancerous cells. Almost all small
molecule anticancer agents are less cancer-specific
than a contemplated halogenated xanthene, and can
result in causing sickness, baldness and other trauma
to their recipient subjects that can lead to subjects
leaving their treatment regimens.
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These small molecules typically have
molecular weights of about 200 to about 1000 Daltons
(Da), and preferably about 250 to about 850 Da. This
group of small molecules includes many of the
previously noted molecules used to treating hematologic
cancers such as calicheamicin (1368 Da), vinblastine
(811 Da), vincristine (825 Da), monomethyl auristatin
(718 Da), etoposide (589 Da), daunorubicin (528 Da),
doxorubicin (544 Da), annamycin (640 Da), sorafenib
(465 Da), clofarabine (304 Da), cisplatin (300 Da),
irinotecan (587 Da) and cytabarine (243 Da). It is
noted that many of these small molecules are used as
their salts, prodrugs and/or esters, which consequently
have greater molecular weights than those rounded
values above.
A pharmaceutical composition having a second
cytotoxic anti-cancer agent_ can also contain a small
molecule as above-described that is conjugated to a
lager molecule such as a protein, detergent and/or
polymer such as poly(ethylene glycol) [PEG]. Such
conjugations often minimize the toxicity of the small
molecule and enhance situs of delivery as use of an
antibody that binds to a cancerous cell. Additionally,
a small molecule cytotoxic agent can be enveloped
within a liposome, micelle or cyclodextrin molecule
that can be adapted to bind specifically bind to
cancerous cells and/or be endocytosed by the cancer
cell. This group of encapsulated and conjugated small
molecules' is included with the previously discussed
small molecule group of second cytotoxic agents as
their active cytotoxic agent is a small molecule.
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Illustrative of such second cytotoxic agents
are liposomal daunorubicin, liposomal annamycin,
sphingosomal vincristine, liposomal cytarabine, a
ealicheamicin-conjugated CD33 antibody called
gemtuzumab ozogamicin and a chimer of CD30 antibody and
monomethyl auristatin E called brentuximab vedotin.
Briefly, liposomes are generally spherically-
shaped artificial vesicles typically prepared from
cholesterol and phospholipid molecules that constitute
one or two bilayers and encapsulate the small molecule
second cytotoxic agent to assist delivery. See,
Akbarzadeh et al., Nanoscale Res Lett, 8:102 (2013).
Calicheamicin, is a high molecular weight
small molecule (1368 Da), and contains four linked
saccharides interrupted by a benzothioate S-ester
linkage as well as an ene-diyne group that cleaves DNA
sequences. Calicheamicin is too toxic: Lo be used
alone, LD50 in nude mice of 320 lig/kg [DiJoseph et al.,
Blood 103:1807-1814 (2004)1. Similarly, monomethyl
auristatin exhibits general (broad range), high
toxicity [IC50 < 1 nM for several cancer cell lines;
ApexBio Technology Product Catalog (2013)] that is
mediated by linkage to an antibody against CD30 (a TNF
receptor-family member that is a cell membrane protein
and cancer marker) was reported useful against large
cell lymphoma and Hodgkin's disease [Francisco et al.,
Blood 102:1458-1465 (2003)], whereas linkage to an
anti-CD79b monoclonal provided an advantage in treating
three xenograft models of NHL [Dornan et al., Blood
114:2721-2729 (2009)1.
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A systemic anti-cancer medication that is a
small molecule (non-proteinaceous, less than about 1000
grams/mole) or a larger proteinaceous molecule, is
administered to the subject mammal to be treated such
that the medication spreads throughout the subject's
body as compared to the localized administration that
occurs with an intralesional administration of a
halogenated xanthene. Intravenous administration is a
preferred method to achieve that spread of medication.
Illustrative small molecule anti-cancer
medications include doxorubicin, etoposide,
vincristine, cisplatin, irinotecan and cytarabine that
were used herein, whereas an exemplary proteinaceous
molecule is egasparaginase. Of those medications,
doxorubicin, etoposide and vincristine appeared to
synergize with treatment with a sub-lethal dose of
PV-10, and are preferred.
It is to be understood that administration of
any of the second cancer cytotoxic agents discussed
herein can be undertaken multiple times. Such multiple
administrations are within the purview of the treating
physician, and can be made in conjunction with an
administration of a first cancer cytotoxic agent or can
be carried out separately.
A useful effective dosage of a small molecule
systemic anti-cancer medication is the dosage set out
in the labeling information of a FDA-, national- or
international agency-approved medication. Typically,
monotherapy dose schedules are set by determining the
maximum tolerated dose (MTD) in early-stage clinical
trials. The MTD (or a close variation thereon) is then
promulgated to later-stage clinical trials for
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assessment efficacy and more detailed assessment of
safety. These MTDs frequently become the established
therapeutic dose upon completion of clinical testing.
However, because the small molecule, systemic anti-
cancer medication is contemplated for use with PV-10, a
MTD is the maximal amount that would normally be used,
and that amount is to be titrated downward following
usual procedures.
Exemplary dosing schedules for a number of
systemic anti-cancer medications that can be combined
with halogenated xanthene therapy in the present
invention are provided in Table A, below. It is noted
that several of the medications listed below are "small
molecules" as defined above, whereas others are large,
proteinaceous molecules such as antibodies. They are
nonetheless administered systemically.
Table A
Exemplary systemic immunomodulatory
or targeted anticancer agents
Systemic Agent Typical Dose
Schedule
adalimumab 80 mg initial
dose followed in 1
week by 40 mg every other week SQ
brodalumab 210 mg
subcutaneously (SC) at Weeks
0, 1, and 2, then 210 mg SC q2wk
certolizumab pegol 400 mg initially and at weeks 2 and
4 followed by 200 mg every other
week or 400 mg Q4 weeks maintenance
SQ
etanercept 50 mg twice
weekly for 3 months
followed by 50 mg once weekly SQ
golimumab 50 mg once a
month SQ
guselkumab 100 mg
subcutaneous injection once
every 8 weeks, after starter doses
at weeks 0 and 4
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infliximab 5 mg/kg given as
an IV induction
regimen at 0, 2, and 6 weeks
followed by a maintenance regimen
of 5 mg/kg every 8 weeks thereafter
ixekizumab 160 mg initial
dose followed Q2
weeks with 80 mg until week 12 then
80 mg QA weeks SQ
sarilumab 200 mg every 2
weeks as a
subcutaneous injection
secukinumab 300 mg every
week for 4 weeks then
300 mg every 4 weeks SQ
ustekinumab Less than 100
kg: 45 mg initially,
week 4 followed by 45 mg every 12
weeks SQ
More than 100 kg: 90 mg initially,
week 4 followed by 90 mg every 12
weeks SO
apremilast Titrated dose
over 5 days to work
up to 30 mg twice daily PO
methotrexate Weekly single
oral, IM or TV 10 to
25 mg per week or divided 2.5 mg
dose at 12 hour intervals for three
doses
cyclosporine Initial dose 2.5
mg/kg/day taken
twice daily as divided (BID); dose
titrated up to 4 mg/kg/day BID if
response and laboratory
abnormalities don't ensue.
azathioprine Used off label
for skin diseases,
1.0 mg/kg oral or IV as a single
dose or twice a day, dose maximum
is 2.5 mg/kg/day.
Because of additive or synergistic effects,
the combination therapies and method of treatment of
the present invention generally permit use of the
systemic agent at a level at or below the typical dose
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schedule for the systemic agent, such as those
described in Table AL, when used with an IV
administration therapy, such as that described below.
However, the dosing schedules provided in Table A
provide a useful guide for beginning treatment from
which dosages can be titrated to lessened amounts as
seen appropriate by the physician caring for a given
patient.
It is noted that a halogenated xanthene and a
second cytotoxic anti-cancer agent need not be
administered together nor by the same means of
administration. Thus, a pill or capsule can be used to
administer the second cytotoxic anti-cancer agent,
while the halogenated xanthene is administered IV.
Those skilled in the art are well aware of the various
methods of administering anticancer agents.
A second type of second cytotoxic agent
useful for combination treatment with a halogenated
xanthene such as that present in PV-10 is an immune
checkpoint inhibitor, that can also be viewed as a
special systemic anti-cancer medication. An immune
checkpoint inhibitor is a drug that binds to and blocks
certain checkpoint proteins made by immune system cells
such as T cells and some cancer cells. When not
blocked, those proteins inhibit immune responses,
helping keep immune responses in check and keeping T
cells from killing cancer cells. Blocking those immune
checkpoint proteins releases the "brakes" on the immune
system permitting T cells to become activated and kill
cancer cells.
A useful immune checkpoint inhibitor is
preferably a human or humanized monoclonal antibody or
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binding portion thereof whose administration blocks the
action of those certain proteins, thereby permitting
the immune system to recognize the cancer cells as
foreign and assist in eliminating those cancer cells
from the body. Illustrative immune checkpoint
inhibitors include the anti-CTLA-4 (cytotoxic T
lymphocyte-associated antigen 4) monoclonal antibodies
ipilimumab and tremelimumab that are designed to
counter down-regulation of the immune system by
blocking CTLA-4 activity and thus augment T-cell
response against cancer. Similarly, monoclonal
antibodies such as pidilizumab, nivolumab,
lambrolizumab and pembrolizumab bind to PD-1
(programmed death 1) receptor to counter down-
regulation of the immune system and augment T-cell
responses to cancerous cells. Three antibodies that
target the immune checkpoint protein ligand (PD-L1) for
the PD-1 receptor (PD-L1) are atezolizumab, avelumab
and durvalumab. Initial work with antibodies to the
PD-1 receptor ligands, PD-Li and PD-L2, such as BMS-
936559 and MEDI4736 (durvalumab) to PD-L1, also
indicate inhibition of down-regulation of the immune
system and an augmented T-cell response against cancer.
Another group of antibodies with checkpoint
inhibitor-like activity immunoreact with the cell
surface receptor 0X40 (C0134) to stimulate
proliferation of memory and effector T-lymphocytes, and
thereby stimulate a T-cell-mediated immune response
against cancerous cells. Exemplary such humanized
anti-0X40 monoclonal antibodies include those presently
referred to in the literature as gsk3174998 (IgG1),
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pogalizumab (M0XR0916), ME1J10562 and the human anti-
0X40 IgG2 antibody designated PF-04518600 (PF-8600).
Intact monoclonal antibodies, as well their
paratope-containing portions (binding site-containing
portions) such as Fab, Fab', F(ab1)2 and Fv regions, as
well as single-stranded peptide binding sequences can
be useful as immune checkpoint protein inhibitors.
Intact checkpoint inhibiting monoclonal antibodies have
half-lives in a human body of about one to three weeks
[e.g., Yervoy (ipilimumab) terminal tin = 15.4 days;
package insert 12/2013; Keytruda0 (pembrolizumab)
terminal t1/2 = 23 days; package insert 03/2017], and
single-stranded oligo or polypeptides tend to have
shorter half-lives in viva.
Because of the relatively short half-lives of
the small molecule second cytotoxic anticancer agents
and a halogenated xanthene medicament, both medicaments
can be administered in a single composition or in
separate compositions. If administered separately, it
is preferred to administer both types of anticancer
agent within minutes to about 3 hours of each other.
More preferably, both are administered within less than
one hour of the other.
As used herein, "administration" is used to
mean the beginning of a treatment regimen. Thus,
swallowing a tablet or other per os dosage form is the
beginning of a treatment regimen, as is the time at
which an IV flow is begun. When both first and second
cytotoxic anticancer agents are present together in the
same, single composition, administration begins when
that unitary composition enters the subject's body.
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Where the second cytotoxic anticancer agent
is an immune checkpoint inhibitor such as a monoclonal
antibody, the halogenated xanthene and the second
cytotoxic anticancer agent immune checkpoint inhibitor
can be administered together or one before the other,
with the second cytotoxic anticancer agent immune
checkpoint inhibitor being administered up to about one
month prior to the halogenated xanthene. Preferably,
the two cytotoxic anticancer agents are administered
together or with the second cytotoxic anticancer agent
immune checkpoint inhibitor being administered within a
few days after the halogenated xanthene. A second
cytotoxic anticancer agent immune checkpoint inhibitor
can also be administered about one month after the
halogenated xanthene.
Results
Results of preliminary in vitro cell culture
viability assays were carried out on a panel of eleven
commercially available leukemia cell lines derived from
patients with either primary or relapsed pediatric leukemia
that were treated with PV-10 (Table 1) and two primary
leukemia samples (Table 2).
Cell viability was measured by alamar blue assay,
96 hours post-treatment. PV-10 decreased cancer cell
viability in a concentration and time dependent manner in the
eleven pediatric leukemia cell lines (mean IC50 92.8 pM), and
three primary leukemia samples (mean IC50 122.5 pM) examined.
The results show that PV-10 is cytotoxic to leukemia
cell lines with a mean IC50 value of 92.8 pM (Table 1,
below) and is cytotoxic to two primary leukemia samples
with a mean IC50 value of 122.5 pM (Table 2, below).
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Table 1*
Cell Line Cell
Type PV-10 IC50
PM
KOPM8 Infant
ALL 150
SUPB15 3-ALL
129
acute T-ALL
121
lymphoblastic
leukemiatib=20
TIB-202 AML
118
SEM B-ALL
99
CCRF-SB B-ALL
88
Kasumil AML
72
MV4-11
Biphenotypic 68
Molm13 AML
42
Molt4 T-ALL
41
Molt3 T-ALL
35
Mean
92.8
*Half maximal inhibitory concentration (IC50)
values for pediatric leukemia cell lines treated with PV-10
for 96 hours.
Table 2**
Cell Type
PV-10 IC50 PM
T-ALL
150
Infant AML
95
Mean
122.5
**Half maximal inhibitory concentration (IC50)
values for primary pediatric leukemia samples treated
with PV-10 for 96 hours.
Observation of four different leukemia cell
lines (Molm-13, MV4-11, SEM, TIB-202) by phase-contrast
and time-lapse video microscopy indicated that PV-10
was cytotoxic and not cytostatic to the cancerous
cells. Quantification of dead cells from time-lapse
video microscopy experiments showed that PV-10 was
cytotoxio in a cell line in a concentration-dependent
manner.
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At 24 hours post-treatment with 100 pM PV-10,
88% of MV4-11 cells, 69% of Molm-13 cells, 27% of TIB-
202 cells and 25% of SEM cells had undergone cell
death. When the concentration of PV-10 was increased
to 200 pM, 100% of MV4-11 and Molm13 cells, 94% of SEM
cells and 60% of TIB-202 cells had undergone cell
death, 24 hours after treatment.
Additionally, observation by time-lapse video
microscopy suggested that cells were dying by
apoptosis, as treatment with PV-10 led to cell
shrinkage. Induction of apoptosis by PV-10 was
confirmed by dose and time dependent PARE cleavage,
detected by western blot. [Swift et al., Blood, 132,
No. Sqppl II 5207 (November 21, 2018).]
These studies provide first evidence in pre-
clinical data for the activity and mechanisms of action
of PV-10 in pediatric leukemia. These data provide the
rationale for additional studies and the formulation of
an early-phase clinical trial for patients with
relapsed and refractory pediatric leukemia.
Method's:
A panel of eleven cell lines derived from
patients with either primary or relapsed pediatric
leukemia (CEM-C1, CCRF-SB, Kasumi-1, KOPN8, Molm-13,
Molt-3, Molt-4, MV4-11, SEM, SUP-315 and TIB-202) and
cells from three primary leukemia patient specimens
(T-ALL, AML, Infant AML) were treated with increasing
concentrations of PV-10 and cell viability was measured
by alamar blue assay, 96 hours post-treatment. Target
modulation and induction of cell death pathways were
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investigated by western blot, phase-contrast microscopy
and time-lapse video microscopy.
Analysis of cell cycle alterations and
induction of apoptosis were measured by flow eytometry.
Combination studies are performed to identify anti-
cancer agents that are synergistic with PV-10 and
animal models of pediatric leukemia used to identify
the activity of PV-10 against pediatric leukemia in
vivo.
Previous Studies
Previous studies have shown that the
halogenated xanthene compounds discussed previously
provide results against cancerous tumor cells that are
similar to those achieved with RB.
Previous Procedures
A. 1 x 104 cells/mL were injected
subcutaneously into BALB/c nude mice. 2-3 Weeks were
required for tumors to grow to treatable volume.
B. 20 L to 40 L of halogenated xanthene
solutions (0.01, 0.001 or 0.1% w/v) was injected until
tumor was completely infiltrated. One could see the
tumor begin to turn red as the agent was injected.
C. Twenty-four hours after injection, the
tumors were illuminated by laser. Laser: Coherent,
Verdi 5W, 532nm CW laser, 200mW/cm2, 100J/ cm2, 4cm2
treatment zone.
D. Confirmation of Specificity by Bioassay:
Color retention of the agent in the tumor 24 hours
after intratumoral injection was determined by
production of photodynamic damage confined to the tumor
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as evidenced by eschar and volume reduction after
illumination. Failure of tumor to reoccur at the
primary site was recorded.
E. Confirmation of Specificity by
Fluorescence: Retention of compounds in the tumors 24
hours after intratumoral delivery was confirmed by
visually observing fluorescence of the halogenated
xanthene agent in the tumor excited by the CW laser.
The 532 nm exciting wavelength was removed using a
Melles Griot filter Part No. 03 FIM 008. Fluorescence
of the compounds was recorded using an Olympus digital
camera (Model 0-300-L).
Results
I Compound I
Results
Amount
(paw %)
Rose Bengal
0.01 24 hours post illumination
3/3 mice eschar production and
full tumor reduction
days post illumination
3/3 mice reoccurrence of tumor
0.001 24 hours post illumination 3/5 eschar production
(1/5 partial)
5 days post illumination
3/3 mice reoccurrence of tumor
Ethyl Red
0.1 24 hours post illumination
2/2 mice eschar production and
full tumor reduction
3 days post illumination
3/3 mice reoccurrence of tumor
Red 3
0.1 24 hours post illumination 0/3 no eschar and no tumor
reduction
PH-12*
0.01 24 hours post illumination
3/3 mice eschar production and
full tumor reduction
7 days post illumination
0/3 mice reoccurrence of tumor
Ethyl-PB**
0.01 24 hours post illumination
2/2 mice eschar production and
full tumor reduction
5 days post illumination
2/2 mice reoccurrence of tumor
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*PH-12 = 2',4',5',7f-tetrabromo-4,5,6,7-
tetraiodoerythrosin prepared and supplied by Molecular Probes
Inc.
**Phloxine B the unesterified parent compound of Ethyl-
phloxine B has been examined previously and is not retained
in tumors.
Each of the patents, patent applications and
articles cited herein is incorporated by reference.
The articles "a" and "an" are used herein to refer to
one or to more than one (i.e., to at least one) of the
grammatical object of the article. Each of the
patents, patent applications and articles cited herein
is incorporated by reference.
The foregoing description and the examples
are intended as illustrative and are not to be taken as
limiting. Still other variations within the spirit and
scope of this invention are possible and will readily
present themselves to those skilled in the art.
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