Note: Descriptions are shown in the official language in which they were submitted.
COMPOSITION AND METHOD FOR
ORAL TREATMENT OF LEUKEMIA
Descri pti on
Field of the Invention
This invention relates to an oral therapeutic
regi men for t reat i ng bl ood ( hematol ogi c) cancers such
as leukemia particularly effecting such treatments i n
chi I dren.
BACKGROUND ART
An adult human has about 7000 white bl ood
cell s per mi crol i ter (IL) of bl ood. Of those whi te
cells, about 65 percent are granul ocytes ( about
4500/ L), about 30 percent are monocyt es ( about
2100/ L), and about five percent are lymphocytes ( about
350/ L) . Geyt on, Text book of Medical Physiology,
Seventh ed. , W. B. Saunders Co., Philadelphia ( 1986).
The above cell number amounts are, of course,
general i zed average val ues, and granul ocyte counts for
normal pat i ents, i . e. , pat i ents free of disease,
typi call y are about 2000 to about 7000 cell s/ L.
Acute I ymphobl ast i c I eukemi a (ALL) i s a
cancer of the I ymphoi d I i ne of bl ood cel I s begi nni ng i n
the bone marrow, and characteri zed by the devel opment
of large numbers of immature lymphocytes
( I ymphobl asts). There are two basic types of this
di sease. One affects B cell s ( B- ALL) , and the other
affects T cells ( T- ALL) . As an acute leukemia, ALL
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progresses rapi dl y and i s typi cal I y fatal wi t hi n weeks
or months if I eft untreated.
ALL occurs i n both chi I dren and adul ts, with
highest rates seen between the ages three and seven
years. About 75 percent of cases occur before the age
of 6, with a secondary rise after the age of 40. The
overall i nci dence of pedi at r i c ALL i n the Uni ted States
dun i ng 2001-2014 was 34.0 cases per 1 mill i on persons
and among all raci al /et hni c groups.
ALL i s typi cal I y treated i ni ti ally with
chemotherapy aimed at bringing about remission.
This
is then foil owed by further chemotherapy typically over
three years. Treatment usual I y al so i ncl udes
i nt rat hecal chemotherapy (spinal cord i nj ect i on),
because systemi c chemotherapy can have limited
penetration i nto the central nervous system and the
central nervous system i s a common site for rel apse of
ALL.
Chronic I ymphocyti c leukemia (CLL) is a type
of cancer i n whi ch the bone marrow makes too many
lymphocytes, part i cul an y B cel Is. Al though it is
general I y consi dered i ncurabl e, CLL progresses sl owl y
i n most cases. CLL treatment consequently focuses on
control I i ng the di sease and its symptoms rather than on
an outright cure. The decision to start CLL treatment
is taken when the person' s symptoms or blood counts
i ndi cate that the di sease has progressed to a poi nt
where it may affect quality of life.
CLL i s pri man i I y a di sease of ol der adul ts,
most commonly occur ri ng i n peopl e over the age of 50,
with a median age of 70 years at the ti me of diagnosis.
Though I ess common, CLL someti mes affects peopl e
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between 30 and 39 years of age. The i nci dence of CLL
increases very qui ckl y with i ncreasi ng age.
Five-year
survi val foil owi ng di agnosi s i s approxi mat el y 83% i n
the United States.
Acute myel ogenous leukemia (AML) begins in
the bone marrow as a di sorder of the hematopoi et i c stem
cell and i s the most common form of I eukemi a i n adul ts.
It occurs i n both chi I dren and adults. Without
treatment, AML can rapidly progress in the body as new
white blood cells conti nue bei ng made.
Chronic myel ogenous leukemia (CML), al so
known as chronic granulocytic leukemia (CGL), al so
starts i n the bone marrow, but it progresses I ess
rapidly than AML. In its early phases, CML is
characterized by I eukocytosi s, the presence of
i ncreased numbers of i mature granul ocytes i n the
per i pheral bl ood, spl enomegal y and anemi a. These
immature granul ocyt es i ncl ude basophi I s, eosi nophi Is,
and neutrophi I s. The i mature granul ocyt es al so
accumul ate i n the bone marrow, spl een, liver, and
occasi onal ly in other ti ssues. Pat i ents present i
ng
with this disease characteri st i cal I y have more than
75, 000 white blood cells per mi crol i ter (jIL) , and the
count can exceed 500, 000/ L.
CML accounts for about 20 percent of all
I eukemi as i n the Uni ted States. About 15 new cases per
mill i on peopl e are reported each year, I eadi ng to about
3,000 to 4,000 new cases per year. The disease is rare
i n humans bel ow age 45, but i nci dence ri ses rapi dl y to
age 65, and remai ns el evated thereafter. The medi an
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life span of pati ents with chronic myel ogenous leukemia
from the ti me of diagnosis is approximately four years.
About 60 to 80 percent of pat i ents with CML
devel op a bl ast cri Si S.
BI ast cri Si s represents a
mani f estati on of acute I eukemi a. The presence of
certai n markers on the bl ast cell s someti mes suggests a
I ymphoi d or i gi n of these cel I s dun i ng the bl ast cri Si S.
Chemotherapeuti c agents used for the
treatment of the bl ast cri Si s are the same as those
used for the treatment of other acute leukemias.
For
exampl e, cytarabi ne and daunorubi ci n, used for the
treatment of acute myel ocyti c I eukemi a, are used to
treat CML bl ast cri si S.
Predni sone and vi ncri sti ne, a
therapeuti c regi me used i n the treatment of acute
I ymphocyti c leukemias, is al so used to treat CML bl ast
crisis.
Nevertheless, these drug therapies of the
bl ast cri Si s stage of CML are even I ess successful than
are the treatments of other acute leukemias.
Cancer i n chi I dren i s rare wi th an i nci dence
of 140- 155 per mill i on ( age <15 years) per year.
Thi s
transl ates to about 1 i n 7,000 chi I dren is diagnosed
with cancer each year.
Despite the rarity of cancer,
mal i gnant neopl asm i s the most common cause of death
after acci dents i n chi I dren aged 5 to 14 years,
accounti ng for 23 percent of mortal i ty.
Survi val from
chi I dhood cancers, many of whi ch were fatal i n the pre-
chemotherapy era, has i ncreased dramatically from 20 to
30 percent i n the 1960s to 62 percent i n the 1970s, and
more recently to 83 percent. Sal etta et al . ,
Trans!
Pedi at r 3(2): 156- 182 (2014).
Leukemi as are the most common chi I dhood
cancers, accounti ng for about 30% of all pedi at ri c
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( ages 1-14) cancer diagnoses. Acute I ymphobl asti c
1 eukemi a (ALL) accounts for about 25 percent of
chi 1 drens' cancers, and acute myel oi d I eukemi a (AML)
accounts for the remai ni ng about 5 percent.
Sal et ta et
al . , Trans! Pedi at r 3( 2) : 156-182 ( 2014)
Current treatments for ALL i ncl ude pegyl at ed
aspargi nase, I i posomal daunorubi ci n, I i posomal
annamyci n, sphi ngosomal vi ncri st i ne, and 1 i posomal
cytarabi ne. For AML, current treatments include the
use of all t rans- ret i noi c acid (ATRA) , arsenic
t ri oxi de, ant hracycl i ne combi ned with ATRA, and
i darubi ci n with high-dose cytarabi ne. Soraf eni b
(multi ki nase i nhi bi tor) i n combination with cl of arabi ne
and cytarabi ne has found success i n a phase 1 study [
Inaba et al . , J CI i n Oncol 29: 3293-3300 ( 2011) ], and a
cal i cheami ci n- conj ugated CD33 anti body, gemt uzumab
ozogami ci n, known commerci al 1 y as Myl otarg , has shown
promi se [ Zwaan et al . , Br J Haematol 148: 768-776
(2010)1.
Al though the survival rate for pediatric
1 eukemi a has great I y i mproved, rel apse i s a maj or cause
of treatment fail ure. Approxi mate! y 15 to 20 percent
of pediatric ALL patients and 30 to 40 percent of AML
pat i ents rel apse, with relapsed ALL i dent i f i ed as the
fourth most common mal i gnancy i n children.
Treatment of relapsed pediatric 1 eukemi a
i ncl udes i nt ensi f i cat i on of chemot her apeut i c regi mens
and use of bone marrow transplantation ( BMT) .
However,
i ncreasi ng the i nt ensi ty of combi nat i on chemot herapi es
and introduction of second-1 i ne drugs is of ten
accompani ed by cumulative toxi city, with margi nal
i ncrement al benef i ts.
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A key component for understandi ng i mmune
system i nteract i ons agai nst pediatric cancers is the
avail ability of an appl i cabl e ani mal model .
Current
xenograf t model s are I i mi ted because they are
established i n severe combi ned i mmunodef i ci ent ( SCI D)
mi ce and so do not provi de i nf ormati on on the
contri but i on of the i mmune system.
Other approaches
such as human hematopoi et i c stem cell reconsti t uti on i n
i mmunocompetent animals are cumbersome, expensive, and
of ten i nt roduce compl ex bi ol ogi cal variables i nto the
syst em.
Recently, a novel xenograf t tumor model was
devel oped i n i mmunocompet ent mi ce by t ol en i zi ng mi ce
fetuses to human t umor cell s [ Basel et al . , Cancer
Lett. 412: 256- 263 ( 2018) ] . This model is advantageous
because it can be used to better describe the complex
i nteracti on between cancerous cell s and the i mmune
system through a xenograf t technique.
One useful anti-cancer agent group for adult
cancerous tumors are the hal ogenated 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 hal ogenated xanthenes, Rose
Bengal di sodi um, ( 4, 5, 6, 7- t et rachl or o- 2' , 4' , 5' , 7' -
tet rai odof I uorescei n di sodi um; RB) has been found to be
part i cul ar I y effective and easi I y utilized.
A sol ut i on of i odi ne- 131 radi ol abel ed RB has
been used cl i ni cal I y to measure liver f unct i on i n
infants [ Yvart et al . , Eur J Nucl Med 6:355- 359
( 1981) ]. PV- ioe, a steri I e 10 percent w/v RB di sodi
um
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sol ut i on i n aqueous 0.9% sodi um chi or i de for i nj ect i on
is a more recent formulation that is manufactured by
Provect us Bi opharmaceut i cal s, I nc. of Knoxvi I I e, TN.
Previ ous studi es have shown that RB or its
salt from PV- 10 aqueous RB di sodi urn solution
accumul at es i n cancerous cel I I ysosomes [Wachter, et
al . , Proceedi ngs of SPI E, Multi photon Microscopy i n the
Bi omedi cal Sci ences I I , Pen i asamy, A. and So, P. T. C.
( eds), Bell i ngham, Washi ngt on: 4620: 143-147 ( 2002) ]
and i nduces cell death in a range of adult cancers [Qi n
et al . , Cel I Death Di s 8: e2584 ( 2017); Toomey et al
PloS ONE 8( 7) : e68561 ( 2013); Koevary et al . , Intl
Physi ol Pat hophysi ol Pharmacol 4( 2) : 99-107 ( 2012);
Thompson et al . , Mel anoma Res 18( 6) :405-411 ( 2008); and
Zamani et al . , J I mmunotoxi c 11( 4) : 367-375 ( 2014) ] .
PV- 108 aqueous RB di sodi urn solution has been
used i n several cl i ni cal t ri al s, both as a si ngl e anti -
cancer agent and i n conj unct i on with monad l onal
anti body anti -cancer agents, where it has been
admi ni stered i nto solid tumor cancers via i nt ral esi onal
( I L) administration. Several of those trials are
di scussed bel ow. Phase I and phase I I cl i ni cal st
udi es
usi ng PV- 10 aqueous RB di sodi urn sol uti on al one as the
cyt ot oxi c agent ill ust rat i vel y reported "adverse events
were predomi nantl y mi I d to moderate and I ocoregi onal to
the treatment site, with no treatment-associated grade
4 or 5 adverse events" [ Thompson et al . , Ann Surg Oncol
22( 7) : 2135-2142 ( 2015) ] , and "Tr eat ment - Emer gent
Adverse Events (TEAEs) were consi stent with est abl i shed
patterns for each drug, pr i nci pal I y Grade 1-2 i nj ect i on
site react i ons at t ri but ed to PV- 108 aqueous RB di sodi urn
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solution and Grade 1-3 immune-mediated reactions
att ri buted to pembrol i zumab, with no si gni f i cant
over I ap or unexpected toxi ci ti es: . . . " [ Agarwal a et
al . , J CH n Oncol 3]( 15) suppl 9559-9559 (May 26,
2019) ] . It thus appears as though RB is toxic to
cancerous cell s, but non- toxi c to non- cancerous cell s.
Because of the often-times very different
behavior of adult tumors from pediatric tumors, it was
not known whether RB and si mi I ar hal ogenated xanthenes
woul d be effective when used agai nst pediatric
cancerous cells and, particularly, pedi at ri c cancerous
hematol ogi c cells.
Prel i mi nary in vitro and xenograf t
st udi es agai nst neurobl astoma cell I i nes i n cell
cul tures to whi ch RB was added al one or i n conj uncti on
with known anti cancer agents, and by i ntral esi onal
injection i nto sol i d tumor xenografts established i n
mi ce, respectively, were reported by one of the present
i nvent ors and co-workers to exhi bit kill i ng of
pediatric cancerous cell s. Swi ft et al . ,
Oncotargets
Ther, 12:1293-1307 ( February 2019) .
I n addi ti on, i nt ral esi onal admi ni strati on of
a hal ogenated xant hene compound into a tumor provi des
the active cytotoxi c agent di rect I y to the tumor at its
hi ghest concent rat i on. I n a presently cont empl at ed
treatment technique discussed below, administration is
often di stant from the target cancerous hematol ogi c
cells, thereby possi bl y di mi ni shi ng the effectiveness
of the canceroci dal hal ogenated xanthene compound
medication ( agent) .
I n a phase I I cl i ni cal t ri al for pat i ents
with refractory met ast at i c mel anoma, i nt r al esi onal
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i nj ect i on of PV- 10 aqueous RB di sodi um sol ut i on
induced tumor regression with an overall response rate
of 51% [Thompson et al . , Ann Surg Oncol 22( 7) : 2135-2142
( 2015) ]. I nt ral esi onal PV- 10 aqueous RB di sodi um
sol ut i on al so demonstrated ef f i cacy i n combi nat i on with
radi ot her apy i n a phase I I cl i ni cal trial for pat i ents
with i n- t ransi t or met ast at i c mel anoma, with an overall
response rate of 86.6% [Foote et al . , J Surg Oncol
115( 7) : 891-897 ( 2017) ].
I n addition to i nduci ng di rect cancer cell
death, PV- 10 aqueous RB di sodi urn sol uti on
i nt r al esi onal admi ni strati on has al so been shown to
induce a tumor-specific immune response in both mouse
studies [ Qi n et al . , Cel I Death Di s 8:e2584 ( 2017) ;
Toomey et al . , PLoS ONE 8(7):e68561 ( 2013); and Liu et
al . , Oncotar get 7( 25): 37893-37905 ( 2016) ] and i n human
cl i ni cal trials [ Li ppey et al . , J Surg Oncol
114( 3) : 380-384 ( 2016); Ross, J Surg Oncol 109(4):314-
319 ( 2104); Li u et al . , PLoS ONE 13( 4): e0196033 ( 2018);
and Basel et al . , Cancer Lett 412: 256-263 ( 2018) ].
I n
muri ne model s of mel anoma, i nt r al esi onal treatment with
PV- 108 aqueous RB di sodi um sol ut i on i nduced necrosis of
mel anoma cell s and a I ocal i zed i ncr ease i n mononucl ear
t umor- i nf iltr at i ng lymphocytes [ Li ppey et al . , J Surg
Oncol 114(3) : 380-384 ( 2016) ] .
It has been suggested that PV- 10 aqueous RB
di sodi urn sol uti on i nduced i mmunogeni c cell death,
rel easi ng tumor anti gens to nearby anti gen- present i ng
cells (APCs) , and facilitated the activating of anti -
tumor T and B cell s. I n a syngenei c muri ne col on
cancer model, injection of cancer cells treated in
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vitro with PV- 10 aqueous RB di sodi um solution into
mi ce with the same tumor resul ted i n slower tumor
growth [ Qi n et al . , Cell Death Di s 8: e2584 ( 2017) ] .
Furthermore, i n syngenei c muri ne mel anoma model s,
combi nation treatment with i nt r al esi onal PV- 10 aqueous
RB di sodi urn sol ut i on and anti - PD-1 anti body del ayed
tumor growth and enhanced T cel I act i vat i on [ Li u et
al . , PLoS ONE 13( 4) : e0196033 ( 2108)].
Parental US Application No. 16/ 688, 319, filed
on November 19, 2019, teaches that hematol ogi c cancer
cell s such as I eukemi a cell s can be successful I y
treated ( ki I I ed) by contact with an aqueous composi ti on
contai ni ng a hal ogenated xanthene, a pharmaceutically
acceptable salt, or a C1-C4 al kyl ester thereof.
Co-
assi gned US Appl i cat i on Seri al No. 17/214, 590, f i I ed on
March 26, 2021, teaches that solid cancerous tumors can
be successfully treated by oral administration of a
hal ogenated xanthene, its I act one, or a
pharmaceut i call y acceptable sal t or ester thereof.
That oral I y admi ni stered medi cament coul d be i n sol i d
or liquid form.
The di scl osure bel ow descri bes the
contemplated invention and provides results of studies
usi ng oral I y- admi ni stered hal ogenated xanthene
compounds such as rose bengal i n the treatment of
pediatric and adult leukemias.
BRIEF SUMMARY OF THE INVENTION
The present i nvent i on contempl ates a method
of t r eat i ng a mammal i an subj ect havi ng I eukemi a.
The
met hod comprises the steps of admi ni steri ng to such a
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mammal i an subj ect a t herapeut i cal I y ef f ect i ve amount of
a hal ogenat ed xant hene ( HX) , a I act one, a
pharmaceut i call y acceptable salt, or a C1-C4 al kyl or
aromatic ester thereof ( col I ect i vel y referred to her ei n
as an "HX compound") as a f i rst I eukemi a cytotoxi c
agent dissolved or dispersed i n a pharmaceutically
acceptable diluent solid or liquid medium. A
contempl at ed admi ni strati on is typically repeated.
A contemplated treatment met hod can al so be
car r i ed out i n con] unct i on with admi ni strati on to that
same mammal i an sub] ect of a second therapeutically
effective amount of a second, differently-acting
systemic leukemia cyt ot oxi c agent dissolved or
dispersed i n a pharmaceutically acceptable medi urn.
The
second systemic leukemia cyt otoxi c agent can be a small
molecule, i oni zi ng radi at i on, or an i nt act anti body or
paratope- contai ni ng anti body portion such as those
protei naceous anti body mol ecul es that i nhi bit
i nf I ammat ory chemoki ne activity or i mmune checkpoi nt
anti bodi es. The f i rst and the second leukemia
cytotoxi c agents can be admi ni stered together i n the
same or different medi um, or i n the same or different
medi urn at different ti mes. The second I eukemi a
cytotoxi c agent can be admi ni stered i n a sol i d tabl et,
capsul e, pi I I or t he I i ke, in a Ii qui d medi um, or as an
intravenous i nj ect i on or i nf usi on.
In one aspect, use of a small-molecule
I eukemi a cytotoxi c agent havi ng a mol ecul ar wei ght of
about 200 to about 1000 Da i s contempl at ed. Compounds
that synergi ze with a HX Compound such as doxorubi ci n,
et oposi de and vi ncr i st i ne are preferred. I nt act
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anti bodies or paratope- contai ni ng anti body port i ons are
a second group of leukemia cytotoxi c agents.
Preferred
among these agents are those referred to as immune
checkpoi nt i nhi bi tors.
[ See, for exampl e, Darvi n et
al . , Exp Mol Nled, 50: 165 ( 2018) . ]
The present i nventi on al so contempl at es use
of a therapeutically effective amount of an HX compound
as a first leukemia cytotoxi c agent dissolved or
dispersed i n a pharmaceutically acceptable aqueous
medi urn for treatment of a mammal i an subj ect havi ng
leukemia, wherein the halogenated xanthene compound ( HX
compound) i s mai ntai ned i n the mammal i an subj ect for a
period of ti me suff i ci ent to i nduce death of leukemia
cell s. In a further embodi ment, the f i rst I eukemi a
cytotoxi c agent HX compound i s rose bengal , a
pharmaceutically acceptable salt, I act one, or C1-C4
al kyl or aromatic ester thereof.
In a still further
embodiment, the HX compound is rose bengal di sodi um
salt.
Further, the typi cal I y treated leukemia cells
are acute B- cel I or T- cel I I ymphobl asti c leukemia
cell s, chronic I ymphocyti c I eukemi a cell s, or acute
myel oi d I eukemi a cell s.
That any cancer, I et al one a sal i d cancerous
tumor of the GI tract I i ke col orectal cancer, coul d be
affected i n any way by an oral I y- admi ni stered HX
compound as di scl osed i n US Appl i cat i on Seri al No.
17/ 214, 590, filed on March 26, 2021, was quite
unexpected because of I ow HX compound bi oavai I ability,
f i rst- pass I asses of the drug, and al so because of the
relatively short circulatory half-time ( about 30
mi nut es) previ ousl y reported for HX compounds such as
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rose bengal (RB) in other contexts. It was thus
unexpected that oral admi ni strati on of rose bengal
di sodi um, an illustrative HX compound, in a
pharmaceuti call y acceptable sal t form di ssol ved i n an
aqueous di I uent, coul d sl ow the progress of col orectal
tumor devel opment i n ani mal s speci all y bred to devel op
col orectal tumors i n the absence of any treatment.
It
was even more unexpected that an orally-delivered
contemplated HX compound could prevent formation of a
col orectal cancerous tumor i n those specially bred
ani mal s.
That an orally-administered effective amount
of an HX compound coul d al so effectively kill I eukemi a
cell s, as di scl osed herei n, was sti I I more unexpected
for the reasons di scussed above, pl us the fact that
leukemia cells are di stri but ed through out the body i n
the blood stream as well as in the bone marrow. Thus,
leukemia cells provide I ower concent rat i ons of more
diffuse targets for the leukemia cytotoxi c HX compound
to "f i nd" and be taken- up than are the cell s of a solid
tumor that are rel at i vel y more concentrated and
di rect I y fed by the tumor's arteries or are contacted
by i nt ral esi onal admi ni strati on di rect I y into the
tumor.
BRIEF DESCRIPTION OF THE FIGURES
I n the drawi ngs formi ng a part of thi s
di scl osure:
Fi g. 1 is a graph showi ng survi val of CB17
SCI D mice from Charles River Laboratories
International, I nc. , treated with oral I y- admi ni stered
rose bengal di sodi um.
Exponent i all y- growi ng SEM cell s
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(2. 5x106 human ALL cells label I ed with GFP) were
injected i nt ravenous! y i nto each ani mal and the
establishment of tumors was monitored. After 4 weeks
to permit the growth of the tumor, mice were randomized
to three groups. Group 1 (n=9 control animals)
received 100 [11_ of PBS given orally twi ce a week for two
weeks. Group 2 ( n=8 treatment Cohort I ani mal s)
recei ved 25 IAL of rose bengal di sodi urn present at 10 %
w/v i n 0.9 % NaCI aqueous sol uti on that was di I uted i n
PBS to a f i nal vol ume of 100 IAL and given oral I y twi ce a
week for 2 weeks. Group 3 ( n=8 treatment Cohort I I
animals) received 12.5 iit of the above 10 % w/v in 0.9 %
NaCI aqueous sol uti on that was di I uted i n PBS to a
f i nal vol ume of 100 tiL and admi ni stered oral I y twi ce a
week for 2 weeks. [vi dence of di sease progressi on was
moni tored i n all ani mal s and survi val was f ol I owed up
to 120 days f ol I owi ng the i ni ti at i on of treatment.
Data are presented as Kapl an-Mei er esti mates.
Exami ni ng the regi on between 50 and 100 days, the I i ne
nearest to the X-axis represents data for the controls,
the mi ddl e I i ne represents data for the Cohort I I
ani mal s, and the topmost I i ne represents data for the
Cohort I ani mal s.
Fig. 2is a log-log plot of data from several
different studies that plots the log of the rose bengal
concent rat i on admi ni st ered ( mol an i ty) versus the I og of
the duration of the HX compound in the subject up to
the ti me of assessi ng solid tumor treatment, and i s
al so present i n an earl i er form i n US Appl i cat i on
Seri al No. 17/214, 590, filed on March 26, 2021.
"I nt r al esi onal Admi ni strati on" represents data present
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i n Thompson et al . , Mel anoma Res 18: 405- 411 ( 2008);
"Swi ft 2018, 2019" are from Swi ft et al . , J CI i n Oncol
36: Suppl ; abstr 10557 ( 2018) and Swift et al
Oncotar gets Ther 12:1293- 1307 ( 2019); "Oral Apcivii n" are
data from the study reported i n US Appl i cat i on Seri al
No. 17/ 214, 590; and "Oral Leukemia" are new data
presented i n the present appl i cat i on.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
I n one aspect, the present i nvent i on
contempl at es an oral I y admi ni st ered pharmaceut i cal
composi ti on for use i n treatment (kill i ng) of I eukemi a
cell s present i n a mammal i an subj ect . A pr i nci pl
e
cytotoxi c agent in that oral pharmaceutical composition
is a hal ogenated xanthene ( HX), the I act one thereof, a
pharmaceutically acceptable salt thereof, or a C1-C4
al kyl or aromatic ester thereof, that are collectively
ref erred to herei n as an "HX compound" that i s present
in a I eukemi a- t reat i ng effective amount. An oral I
y
admi ni stered pharmaceut i cal composi ti on can be i n solid
or liquid form.
A contempl at ed hal ogenated xanthene mol ecul e
i ncl udes rose bengal ( 4, 5,6, 7- t et rachl oro-2' , 4' , 5' , 7' -
tet rai odof I uorescei n; RB) that is particularly
preferred, eryt hrosi n B, phi oxi ne B, 4, 5, 6, 7-
t et rabromo- 2' ,4' , 5' , 7' - t et r a- i odof I uorescei n,
2', 4, 5, 6, 7- pent achl oro-4' , 5' , 7' - t ri i odof I uorescei n,
4, 4' , 5, 6, 7- pent achl oro- 2' , 5' , 7' -tri i odof I uorescei n,
2' , 4, 5, 6, 7, 7'- hexachl oro- 4' , 5' - di i odof I uorescei n,
4, 4' , 5, 5' , 6, 7- hexachl oro- 2' , 7' - di i odof I uorescei n,
2' , 4, 5, 5' , 6, 7- hexachl oro- 4' , 7' - di i odof I uorescei n,
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4, 5, 6, 7- t et rachl or o- 2' , 4', 5' -trii odof I uorescei n,
4, 5, 6, 7- t et rachl or o- 2' , 4', 7' - t r i i odof I uorescei n,
4, 5, 6, 7- t et rabromo- 2', 4', 5'- tri i odof I uorescei n, and
4, 5,6, 7- t et rabromo- 2' ,4' , 7'- tri i odof I uorescei n.
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
pharmaceuti cal I y acceptable salts with pharmaceuti cal
compounds, such as the above halogenated xanthenes.
Ill ust rat i ve cat i ons i ncl ude al kal i metals such as
sodi um, pot assi um, as well as ammoni um and al kal i ne
earth salts such as magnesi um and cal ci um. The
di sodi um sal t of rose bengal i s part i cul arl y preferred.
The I act one form of a contempl ated
halogenated xanthene can be formed synthetically and is
a preferred precursor of very pure rose bengal . I
n
addi ti on, the carboxyl i c aci d form of a hal ogenated
xanthene salt spontaneously forms the I actone form when
in a strongly acidic aqueous environment such as that
present i n a mammal i an stomach. When formed i n a
mammal i an stomach or si mi I ar I y aci di c aqueous medi urn
from the carboxyl i c acid or carboxyl ate salt form, the
I act one not only forms, but al so appears to aggregate
into clumps that do not readily dissolve in the
duodenum and ad] acent small i nt est i nal regi on or i n an
aqueous medi um havi ng a duodenal pH val ue.
A C1-C4 al kyl ester of one of the above
halogenated xanthene compounds can al so be used, with
the C2; i . e. , ethyl ester, bei ng preferred. In
vitro
studies using each of RB, ethyl -Red 3 (erythrosi ne
ethyl ester; 2' , 4' , 5' , 7' - t et rai odo- f I uorescei n ethyl
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ester) , 4, 5, 6, 7- t et rabromo- 2' , 4' , 5' , 7' - tet rai odo-
f I uorescei n, and ethyl - Phl oxi ne B ( 4, 5,6, 7- t et rachl or o-
2' , 4' , 5' , 7' - t et rabromo- f I uor escei n ethyl ester) exhi bit
Si mi I ar anti -tumor activities agai nst CCL- 142 renal
adenocarci noma.
A contemplated aromatic ester is formed by a
reaction between an HX molecule and an aromatic alcohol
havi ng a 5- or 6- membered aromati c ri ng ( i ncl udi ng
benzyl al cohol ), or a 5, 6- or 6, 6- f used aromat ic ri ng
system that contai ns 0, 1 or 2 het ero ri ng atoms that
are independently nitrogen, oxygen or sulfur. When an
aromatic ester is used, it is preferably a benzyl ,
phenyl , or a 2-, 3-, or 4- pyri dyl ( pyri dyl ) ester,
other aromat i c single and fused ring-containing esters
are contemplated as discussed hereinafter. It is to
be
understood that although a benzyl ester is of ten
consi dered to be an "aral kyl ester", for the purposes
of t hi s i nventi on, a benzyl ester is deemed an aromatic
ester.
I I I ust rat i ve examples of such aromatic
alcohol ester port i ons are shown and named bel ow, where
0 is an oxygen atom and I i ne-0 indicates the ri ng-
oxygen can be from any avail abl e carbon of the ri ng and
the 0-li ne crossed by a wavy I i ne indicates that the
depi cted al koxy group is a portion of another mol ecul e,
the ester i f i ed HX molecule.
c\-535.
sV. ,s
c-r\ )
N N
thienyl furyl oxazolyl thiazolyl
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csS"5
¨ ¨0
naphthyl quinolyl
quinoxalinyl
>
benzofuranyl benzo[b]thienyl benzoxazinyl
Rose bengal i s a preferred HX mol ecul e and
its di sodi um sal t, rose bengal di sodi um, i s a most
pref erred HX compound. A structural f ormul a of rose
bengal di sodi um i s shown bel ow:
CI COONa
Na0 0 0
Further detail s of the medi ci nal use of a
pharmaceutical composition containing an above-noted HX
compound are descri bed i n 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
di scl osures are i ncorporated by reference herei n i n
thei r ent i reti es.
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Dosing - Fig. 2
Upon exposure of tumor cell s in a 0.9 %
sodi urn chl or i de- contai ni ng aqueous medi urn to an HX
compound, i rreversi bl e accumul at i on of the HX compound
occurs i n tumor I ysosomes, causi ng i mmunogeni c tumor
autol ysi s once a suff i ci ent concent rat i on i s achi eyed
to destabilize I ysosomal i ntegri ty [Wachter et al . ,
SPI E 4620: 143-147 ( 2002) ] . Thi s suggests that t hi s
i mmunogeni c mechani sm of cel I death can be el i cited
over a range of exposure conditions based on a
( concent rat i on) = (time f unct i on) , where cyt ot oxi city
is proport i onal to the product of these two parameters
[ i . e. , cytot oxi city = f( [ HX] =t) , where "t" is time].
For exampl e, when RB i s admi ni stered in vi vo
by i nt ral esi onal i nj ect i on to a range of sol i d tumors
( e. g. , melanoma, hepatocel I ul ar carci noma, breast
carci noma) acute tumor cytot oxi city i s evi dent wi t hi n
approximately 30 mi flutes for i nt rat umoral RB
concent rat i ons of approximately 25-50 mg/ g tumor tissue
( 25-50 mM) [ Thompson et al, Melanoma Res 18: 405-411
( 2008) ].
Swift et al . [Oncotargets Ther 12:1293-1307
( 2019) ] demonstrated cytotoxi city of treatment-
ref ractory pediatric solid tumors (neurobl astoma and
neuroepi t hel i oma) upon in vitro contact with RB for 96
hours at concent rat i ons of approximately 50-100 M.
Addi ti onal I y, Swi ft et al . , [J CI i n Oncol 36: Suppl ;
abst r 10557 ( 2018) ] , showed cyt ot oxi city in addi ti onal
treatment- refractory pedi at r i c solid tumors ( Ewi ng
sarcoma, osteosarcoma and rhabdomyosarcoma) under
equi val ent exposure.
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Extended exposure to RB i n the context of
conti nuous oral f eedi ng has been shown to prevent
format i on of col on cancer ( prophyl act i c act i vi ty) and
to arrest col on cancer (therapeutic activity) i n the
muri ne ApcMi n col orect al tumor model as di scl osed i n
parental US Appl i cat i on Seri al No. 17/214590, f i I ed on
March 26, 2021. For t herapeuti c use, symptomatic mi ce
recei vi ng RB ad Ii bi turn i n dri nki ng water at a
concent rat i on of 1 mg/ mL had an approxi mate 38%
increase i n mean survi val relative to untreated mi ce
( 12. 3 O. 5 weeks vs 9. 8 O. 8 weeks) . Presumi ng a
daily dr i nki ng water consumpti on rate of approxi mate! y
2 mL/ 10 g body wei ght, this corresponds to consumption
of approximately 2 mg RB/l0 g ( 200 mg/ kg) .
Bi oavai I ability of RB di sodi um admi ni stered
in aqueous solution via the oral route appears to be
limited based on mass balance studies conducted by the
inventors, and can be esti mated at O. 1- 1 percent,
correspondi ng to a dai I y systemi c exposure of O. 2-2
mg/ kg. Presumi ng this amount is distributed through
the bl oodst ream, and that bl ood vol ume comprises
approxi mate! y 10 percent of body wei ght, t hi s equates
to an esti mated concent rat i on of 2-20 M RB in the
bl ood.
Thi s same approach was used to pl ot data
presented i n Fi g. 1 of the present appl i cat i on, which
shows survi val of CB17 SCI D mi ce with establ i shed
xenografts of a pedi at ric B acute I ymphobl asti c
leukemia (ALL) tumor cel I I i ne; therapeutic act i vi ty
was observed for mi ce i n two treatment groups recei vi ng
RB by gavage twice weekly for two consecutive weeks.
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Assuming 1% bi oavai lability of this oral RB, an
i nt est i nal transit ti me of 6 hours per admi ni strati on,
and a bl ood vol ume of approxi mate! y 10 percent of body
weight, the two treatment groups correspond to an
esti mated 125-250 pril RB i n the blood.
PI otti ng these data conf i rm that the
hypothesized relationship ( i . e. , cytotoxi city =
f( [ HX] =t)) is supported by experimental results, as
illustrated in Fig. 2.
More importantly, this functional
rel at i onshi p permits predi ct i on of dose level and
schedule appropriate to achi eve either an anti-tumor
t her apeut i c outcome upon systemi c admi ni strati on.
For
extended systemic treatment schedules equivalent to
that i nvesti gated with the ApcMi n model, low mi cromol ar
concent rat i ons ( i . e. , about 10 M) of ci rcul at i ng HX
compound are sufficient to achi eve I ysosomal
accumulation and tumor cell destruction over a period
of approxi mate! y 3 months, whereas mi cromol ar to
submi cromol ar concent rat i ons ( i . e. , about 1 M) are
suff i ci ent to achi eve tumor cel I destruction over a
pen i od of approxi matel y 12 months.
Conversely, shorter durati on or i nterrupted
repeat systemi c dosi ng at hi gher dose I evel , as used i n
the present oral I eukemi a model , can al so achi eve tumor
dest r uct i on.
For a speci fic i ndi cat i on, such as treatment
of pedi at ri c pat i ents with I eukemi a, the rel at i onshi p
of Fi g. 2 ill ust rates that standard approaches
rout i nel y used by those of ski II in the art in
pharmaceutical development can be applied to select an
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appropri ate dose I evel and schedul e that maxi mi zes
therapeuti c outcome whi I e mi ni mi zi ng potent i al safety
ri sk.
The ApcMi n data of Application Seri al No.
17/214590 and the oral I eukemi a treatment data of the
present appl i cat i on show that a si mpl e f ormul at i on of
the di sodi um salt of RB is suff i ci ent to deliver a
therapeutically active level of RB; however, this may
be I ess than ideally eff i ci ent as to bi oavai lability.
Determi ni ng a suitable f ormul at i on to achi eve eff i ci ent
liberation and absorption of an orally delivered HX
compound i s thus a matter of standard pharmaceuti cal
devel opment fami I i ar to those of ski II in the art,
where the properties of the formulation can be varied
to achi eve desi red bi oavai I ability by control of
liberation ( di si ntegrat i on, di saggregati on and
di ssol uti on) at an appropri ate poi nt wi thi n the GI
tract so as to maximize absorption of the dissolved HX
compound i nto the bloodstream.
Formulary optimization can be guided by
standard pharmacoki net i c study of absorption such that
dose I evel and formul at i on are ad] usted to achi eve the
necessary systemi c exposure on the desi red dose
schedule ( e. g. , about 100 1.011 in the bloodstream for
short durati on exposure on the order of several days,
about 1 to about 10 IP for i ntermedi ate durati on
exposure on the order of several months, to about < 1
plvl or lower for long-term exposure on the order of a
year or more).
The di basic salt forms of the HX compounds
exi st i n sol uti on havi ng a pH greater than
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approxi mate! y 5, whereas at pH val ues < 5 the HX
compounds spontaneously convert to their I actone form.
Because the di basi c salt forms are hi ghl y sol ubl e i n
aqueous medi a, whereas the 1 act one forms are i nsol ubl e
in aqueous media, the former exhibit hi gher
bi oavai 1 ability i n the GI tract compared to the latter.
Thus, opt i mi zi ng f ormul at i on to properly compensate for
the pH val ue of the GI tract i s perhaps the most
important parameter aff ecti ng bi oavai I ability. For
exampl e, i n the stomach, where pH val ue < 4, di ssol ved
HX compound is rapidly converted to the insoluble
1 actone form. Once i n the 1 act one form, the HX
compounds exhi bit hysteresi s and hi nderance to
saponification back to the absorbable salt form,
del ayi ng or i nhi bi ti ng downstream bi oavai lability.
However, the i ntral umi nal pH val ue rapi dl y
i ncreases from hi ghl y aci di c in the stomach to around
pH 6 i n the duodenum, and further i ncreases i n the
small i nt est i ne from pH 6 to about pH 7.4 i n the
termi nal ileum; pH drops to 5.7 i n the caecum before
gradual I y i ncreasi ng to pH 6.7 i n the rectum.
[ pubmed. ncbi . nl m. ni h. govt 10421978/. 1 Thus, by appl yi ng
standard means in the art of pharmaceutical formulation
to achi eve i nt est i nal I i berati on, where the f avorabl e
pH val ues facilitate HX compound I i berati on i n a
di ssol vabl e, absorbabl e di basi c sal t form,
bi oavai I abi I i ty is optimized.
For a 12-month treatment regi men, these data
i ndi cate that a target concent rat i on of approxi mate! y 1
iiM ( 1 mg/ L) i s achi eyed i n the bl oodst ream. For a
70
kg adul t human, where bl ood vol ume compri ses
approximately 10% of body weight ( i . e. , about 7 L),
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this implies absorption of 7 mg HX compound/ day.
If
bi oavai lability is limited to 1% of admi ni stered HX
compound, then 700 mg HX compound per os (P0) woul d be
requi red daily to achi eve t hi s target bl ood I evel .
However, by opt i mi zi ng absorpti on to 50% of
admi ni stered dose, the necessary PO dose i s reduced to
approximately 15 mg daily. For a shorter treatment
regi men ( i . e. , 3 months), these data i ndi cat e that a
target blood concentration of approximately 10 M ( 10
mg/ L) is achi eyed.
Presumi ng 1% bi oavai lability, then
7 g HX compound PO i s requi red dai I y, whereas at 50%
bi oavai lability, the necessary dose i s reduced to
approxi mate! y 150 mg dai I y.
One contempl at ed pharmaceutical composition
compri ses a O. 1 % to about 20 % (w/v) aqueous medi um
(as a I i qui d) of a f i rst leukemia cytotoxi c agent that
is a halogenated xanthene compound ( HX compound). More
preferably, that concent rat i on is about 0.2 to about 10
'Yo ( w/v), most preferably, the concent rat i on is about
0.2 to about 5 % ( w/v). Thus, for example, the above
dose of 150 mg dai I y coul d readily be achi eyed by use
of 3 mL of a 5 % ( w/v) aqueous solution.
A part i cul ar I y preferred halogenated xant hene
salt is rose bengal ( 4, 5, 6, 7- t et rachl oro- 2' , 4' , 5', 7' -
t et rai odof I uorescei n) di sodi um ( RB di sodi um) salt.
The
pharmaceuti cal composi ti on i s admi ni stered oral I y to
provi de a therapeutically effective amount of a f i rst
leukemia cytotoxi c agent to a mammal such as a human
havi ng leukemia, or more specifically, acute
I ymphobl asti c I eukemi a (ALL) as T- ALL or B- ALL, chronic
I ymphocyti c leukemia (CLL), or acute myeloid leukemia
(AML).
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The mammal i an subj ect i s typically treated
mul ti pl e ti mes. The fact and relative amount of
I eukemi a cell kill i ng can be determi ned by usual means
for assayi ng the status of a gi ven I eukemi a mammal i an
subjects. Both the duration of maintenance and the
choice to conduct further admi ni strati ons can depend
upon the speci es of mammal , i ndi vi dual mammal i an
subjects, the severity of disease, type of disease, age
and health of the subject, and the observed effect on
the burden of leukemic cells caused by the treatment.
These factors are commonly dealt with by physicians
ski I I ed i n the art of t reat i ng leukemia.
I n addi t i on, whereas it is typi cal I y desi red
to rid the body of detect abl e I eukemi c cell s, that
cannot al ways be done. Sometimes it is sufficient to
kill enough I eukemi c cel Is to control the di sease i n
stasis, or to reduce the leukemic load of cells so that
other therapies can be carried out.
The data provi ded herei naf ter ill ust rate that
the I Cso val ue for use of RB agai nst several I eukemi a
cell lines in vitro is about 50 to about 100 pLM for
exposures of one to several days. Given that the
molecular weight of RB di sodi um is 1018 g/mol e, the
above I Cso value calculates to about 50 to about 100 mg
of RB/ I i ter. It is preferred to achi eve that
concent rat i on for contact i ng I eukemi c cells dun i ng an
in vivo treatment.
The cl assi c i nt ravenous ( I V) di agnost i c assay
for liver function usi ng RB was conducted gi vi ng 100 mg
RB as a single IV dose.
In clinical studies of PV- 10
aqueous RB di sodi um sol ut i on, RB has been t ol erated at
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1500 mg deli vered IV. The standard adul t bl ood vol ume
is approximately 5 L.
Thus, to achieve 100 mg/ Lint he
blood, an adult patient would need to receive
approxi mate! y 500 mg of RB IV to achi eve the I C50 val ue
in the bloodstream.
Due to the rapid clearance of RB
from circulation ( t112 is about 30 minutes), an IV
admi ni strati on can requi re cont i nuous i nf usi on to
mai ntai n peak I evel s of RB i n ci rcul at i on ( i . e. , for up
to several hours or more) .
Administration at the I C50 val ue level would
not be toxi c to all ci rcul at i ng hematol ogi c, non-
tumorous I eukemi c cell s; i . e. , onl y approxi matel y hal f
of cells would be affected at the I C50 val ue.
It can
therefore be preferred to admi ni ster RB at a multi pl e
of the I C50 value, up to approximately 1500 mg ( i . e. ,
300 tiM).
Alternatively, it can be suf f i ci ent to kill
only a f ract i on of the I eukemi c cells to initiate a
f uncti onal i mmune response agai nst remai ni ng I eukemi c
burden. The latter case can be preferable to avoid
toxi c react i on ( i . e. , so- cal led "tumor I ysi s syndrome")
due to presence of an abundance of rapidly killed
leukemia cells. I n t hi s Si tuat i on, the I eukemi a
cell
debri s caused by the cytotoxi city to I eukemi a cell s of
a hal ogenated xant hene rel eases i ntracel I ul ar contents
such as pot assi um, causi ng non- speci f i c cell death.
This process may al so activate the immune system
specifically agai nst the mal i gnant cells.
The similarly useful hal ogenated xanthene
compounds previ ousl y- I i sted and thei r pharmaceuti cal I y
acceptable salts can have molecular weights that differ
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from each other by about a factor of three ( See, Table
3, US Patent No. 7, 390, 688 at col umns 15- 16) . It is
preferred that an exact amount of other than RB
halogenated xanthene to be used is calculated based on
publ i shed mol ecul ar wei ghts for each such compound and
that of RB or RB di sodi um.
A mammal i an subj ect havi ng I eukemi a i n need
of treatment (a mammal i an subj ect) and to which a
pharmaceutical composition containing a halogenated
xanthene compound can be admi ni stered can be a pri mate
such as a human, an ape such as a chi mpanzee or
gori I I a, a monkey such as a cynomol gus monkey or a
macaque, a I aboratory ani mal such as a rat, mouse or
rabbit, a companion ani mal such as a dog, cat, horse,
or a food ani mal such as a cow or steer, sheep, I amb,
pig, goat, llama or the like.
I n one aspect of the i nventi on, a
contempl ated HX compound for oral admi ni strati on i s
typi call y used di ssol ved or di spersed i n a steri I e
aqueous pharmaceutical composition.
Sterile tap water
or sterile water from another source can be used.
Characteristics of a Contemplated Liquid Pharmaceutical
Composi ti on
An HX compound i s typi call y present i n a
contempl ated aqueous pharmaceut i cal composition at
about 0.1 to about 20 % ( w/v). More preferably, that
concent rat i on i s about O. 2 to about 10 % (w/v), most
preferabl y, the concent rat i on i s about 0.2 to about 5 %
(w/v). Thus, for example, the above dose of 150 mg
dai I y coul d readi I y be achi eyed by use of 3 mL of a 5 %
(w/ v) aqueous sol ut i on.
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The bi oavai I ability of van i ous HX compounds
such as di sodi urn rose bengal has not been well
characterized. Studies commissioned by one of the
assi gnees concl uded that bi oavai I ability of di sodi urn
rose bengal is less than (<) 1% based on radi ol abel
studies where 14C- RB in aqueous solution was given
oral I y to mi ce.
I n the stomach, with a pH val ue < 4,
an HX compound is Ii kel y to be i n the I actone form.
Conversi on to I act one i n the stomach does not destroy
the HX compound, but such conversi on i nto the I act one
form can present a ki net i c and/or thermodynamic barrier
to reconversi on to the sol ubl e sal t form necessary for
absorpti on i n the i nt est i nes.
The study di scussed i n U.S. Patent
Application Seri al No. 17/214590 using Apcmi n mi ce
showed that those mi ce consumed 4 mg/ mL ad Ii bi turn i n
dri nki ng water arrested onset of disease. Those Apcmi n
mi ce are understood to have thereby consumed
approxi mate! y 8 mg/ 10 g/ day = 800 mg/ kg/ day. That
amount i s consi stent with toxi col ogy data showi ng such
a dose i s t ol erat ed.
Thus, I to et al . , J Nat! Cancer
1, 77: 277- 281 ( 1986) studying rose bengal as a food
col or i ng ( Food Red No. 105) found that rose bengal fed
ad libitum continuously for 2 years at a dose of 970
mg/ kg/ day to C57BL6N mi ce was well tolerated. The
previ ousl y used i nt ravenous (I V) I i ver di agnosti c
deli vered 112 mg of rose bengal as a bol us; for a
standard 60 kg adult human, which equates to 1.9 mg/ kg;
this has not reported to yield morbidity.
It is preferred that a liquid pharmaceuti cal
composi ti on for oral admi ni strati on have an osmol al i ty
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less than that of blood plasma.
Normal (well) human
reference range of osmol al i ty i n pl asma i s about 275-
299 mill i - osmol es per ki I ogr am (mOsm/ kg).
More preferably, that composition is free of
toni city agents ( or toni city-adjusting agents) such as
sugars I i ke manni tol and dextrose, C3-C6 pol yhydroxy
compounds such as propyl ene gl ycol , gl ycerol and
sorbi tol , i sotoni c salts such as sodi um or potassi um
chl on de, and/or buff eri ng agents other than those such
as citric acid, mal i c acid, acetic acid and other food
aci ds and thei r salts that can be provi ded for fl avor
and mild buffering (less than 5 mmol of buffering
agent) . The stomach and I ower GI tract are well
adapted to provi de the proper toni ci ty to materi al s
fl owi ng through such that further salts and/or buffers
are not needed. One or more pharmaceuti cal I y
acceptable taste- maski ng agents or fl avorants as are
well - known can be present at up to about 5% by wei ght
to enhance the potability of the composi ti on.
It is preferred that the pH val ue of a
pharmaceuti call y acceptable aqueous di I uent be about 5
to about 9, to yield maxi mum sol ubi I i ty of the HX
compound i n an aqueous vehi cl e and assure compati bi I i ty
with biological ti ssue.
A particularly preferred pH
val ue is about 5 to about 8, and more preferably
between about 6 to about 7.5. At these pH val ues, the
hal ogenated xanthenes typi call y remai n i n di basi c form,
rather than the I actone that forms at low pH val ues.
An HX compound such as rose bengal i s
di basi c, havi ng pKa val ues of 2.52 and 1.81.
pKa val ue
determi nati ons for several contempl ated hal ogenated
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CA 03175637 2022- 10- 14
xant henes can be found i n Batsi tel a et al . , Spectrochi m
Acta Part A 79( 5) : 889-897 (Sept. 2011) .
I n the present i nvent i on, the specific amount
of halogenated xanthene compound in a pharmaceutical
composi ti on i s not bel i eyed to be as i mport ant as was
the case where the composition was injected
i nt ral esi onal ly to a tumor because the object here is
to ul ti matel y provide a cytotoxi c concent rat i on of
hal ogenated xanthene compound to the envi ronment of the
I eukemi c cell s and i n whi ch those I eukemi c cell s can be
contacted with the halogenated xanthene compound. The
data provi ded herei nafter i ndi cat e that an I CH
concent rat i on of di sodi um rose bengal is about 50 to
about 100 M for in vitro cultured leukemia cells.
The above-noted results using in vitro
cul t ured I eukemi a cell s surpri si ngl y provided data
si mi I ar to those obtai ned i n an in vitro cyt otoxi city
study of cul t ured SK- N- AS, SK- N- BE( 2) , I MR5, LAN1,
SHEP, and SK- N- SH neurobl ast oma cells, SK- N- MC
neuroepi t hel i oma cell s, and normal pr i mary, Bj , and
WI 38 f i brobl asts reported by Swift et al . , OncoTargets
and Therapy 12: 1-15 ( 2019). Those authors reported
half maxi mal inhibitory concentration ( I C50) val ues for
PV- 100 aqueous RB di sodi um sol ut i on-treated cells at 96
hours post treatment of 65-85 M for the neurobl astoma
I i nes assayed and 49 M for the neuroepi thel i oma I i ne
SK- N- MC. Those authors al so examined toxicity toward
human epithelial cells from three tissue sources and
reported I C50 values of 93-143 M.
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I n ci i ni cal studi es of PV- 10 aqueous RB
di sodi um sol uti on, RB has been tol erated at 1500 mg
delivered I L. Due to the rapi d ci earance of RB from
ci rcul at i on (t112 about 30 mi nutes) an IV
admi ni strati on can requi re cont i nuous i nf usi on to
mai ntai n peak I evel s of RB i n ci rcul at i on ( i . e. , for up
to several hours or more) dun i ng a si ngl e
admi ni strati on.
Characteristics of a Contemplated Solid Pharmaceutical
Composi ti on
It is further contempl ated that the HX
compound such as RB or di sodi um RB, or a HX compound
I act one such as RB I act one be admi ni stered i n a sol i d
pharmaceuti cal composi ti on for oral admi ni strati on that
is enteri cal I y- coated to pass through the stomach and
rel ease the HX compound i n the i ntesti nes. The HX
compound i s typi call y di ssol ved i n or di spersed i n or
on a sol i d di I uent medi um.
There are several factors at play in the
di ssol uti on of an oral I y admi ni stered sol i d
pharmaceuti cal product i n a mammal i an body. Among
those factors are resi dence ti me of the medi cament at
different locations along the GI tract, particle size,
sol ubi I i ty of the i ndi vi dual components of the
medi cament i n the bodily fl ui ds I i kel y to be
encountered from mouth to anus, the order i n which
van i ous coati ng layers, when present, are applied to
the medi cament, as well as the pH val ue at whi ch a
part i cul ar coati ng layer is sol ubl e.
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CA 03175637 2022- 10- 14
For example, the highly aci di c gastri c
envi ronment ( pH 1.5-2 in the fasted state; pH 3-6 in
the fed state) ri ses rapi dl y to about pH 6 i n the
duodenum and i ncreases al ong the smal I i ntesti ne to pH
7.4 at the termi nal i I eum. The pH value in the cecum
drops j ust bel ow pH 6 and agai n ri ses i n the col on
reachi ng pH 6. 7 at the rectum [ Hua, Front Pharmacol
11: Art i cl e 524 (April 2020)].
Observation of sol uti ons
of di sodi urn RB mixed i nto a water sol uti on havi ng the
pH val ue of the human stomach reveal ed rapi d cl oudi ng
of the admi xture and cl umpi ng of the previously sol ubl e
di sodi um RB, presumably i nto the I act one form.
Gastric transit can range from 0 to 2 hours
i n the fasted state and can be prolonged up to 6 hours
in the fed state. I n general, the transit time i n the
small i nt est i ne is consi dered relatively constant at
around 3 to 4 hours, but can range from 2 to 6 hours i n
heal thy i ndi vi dual s. Col oni c transit times can be
highly variable, with ranges from 6 to 70 hours
reported [ Hua, Front Pharmacol 11: Art i cl e 524 (April
2020) ] .
Drugs must pass or permeate through the
epi t hel i al cel I s t hat I i ne t he i nner wal I s of t he GI
tract i n order to be absorbed i nto the ci rcul atory
system.
A cell ul ar barni er that can prevent epi t hel i al
cell absorption of a given drug is the cell membrane.
Cell membranes are essentially lipid bi layers that form
a semi per meabl e membrane.
Pure I i pi d bi I ayers are general I y permeabl e
onl y to small , uncharged sol ut es. Hence, whether or not
a mol ecul e is i oni zed will affect its absorpti on,
because i oni c mol ecul es are charged.
Sol ubi I i ty favors
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charged speci es, and permeabi I i ty favors neutral
speci es.
Typi call y, i ons cannot passively diffuse
through the gastroi ntesti nal tract because the
epi thel i al cell membrane is made up of a phosphol i pi d
bi I ayer.
The bi I ayer i s made up of two layers of
phosphol i pi ds i n which the charged hydrophi I i c heads
face outwards and the non-charged hydrophobi c fatty
acid chai ns are i n the mi ddl e of the layer. The
uncharged fatty aci d chai ns repel i oni zed, charged
molecules.
This means that the ionized molecules
cannot easi I y pass through the i ntesti nal membrane and
be absorbed.
Chemi cal modification by est er i f i cat i on can
be used to control sol ubi I i ty. For example, C2-C4
al kyl and aromati c ester forms of an HX compound
typi call y have decreased sol ubi I i ty i n aqueous I i qui ds,
and because of thei r neutral i oni c charge, are
typi call y better taken- up by i ntesti nal epi thel i al
cells than thei r carboxyl ate forms. Later, esterases
i n the GI tract wall and bl ood hydrol yze these esters
to release the parent drug.
Al so, coati ng f i I ms on a tab! et or a pel I et
can act as a barn i er to reduce the rate of di ssol uti on
and/or di si ntegrat i on of the composi ti on in aqueous
media, generally, and particularly within the stomach.
A coati ng can al so be used to modify where di ssol uti on
takes pl ace. For exampl e, enteri c coati ngs can be
appl i ed to a drug- contai ni ng medi cament, so that t he
coati ng and the drug only di ssol ve i n the basic
envi ronment of the i ntesti nes. One approach useful
for
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predictable rel ease of a drug from a medicament in the
i ntesti nal portion of the GI tract and/or at a
part i cul ar location i n the GI tract relies upon pH-
specific coati ngs and mat ri ces that dissolve or
di si ntegrate at presel ected GI tract pH val ues such as
those noted previously.
The tabl e bel ow shows some exampl es of pH-
dependent pol ymer coati ngs that have been used for the
purpose of targeting rel ease (local treatment) either
al one or i n combi nati on, i ncl udi ng some met hacryl i c
resins (commercially available from Evoni k Industries,
AG, Essen, Germany as Eudragi tO), and hydroxypropyl
methyl cel I ul ose ( HPMC; available from DuPont,
Wilmington, DE as Met hocel TM; and Ashl and, I nc. , as
Benecel TN, W I I mi ngt on, DE) der i vat i yes.
I n addi ti on to
tri ggeri ng rel ease at a speci f i c pH value range, the
enteri c coati ng can protect the incorporated active
agent agai nst the harsh GI tract envi ronment ( e. g. ,
gastri c j ui ce, bi I e aci d, and mi crobi al degradati on)
and can create an extended and del ayed drug rel ease
prof i I e to enhance therapeutic eff i ci ency.
The "publ i shed pH rel ease" val ue for each
pol ymer is from the manufacturer. The "publ i shed pH
rel ease" val ues are not absol ute for all composi ti ons
or envi ronments, and pH val ues for di ssol uti on or
di si ntegrati on stated herei n are based on those
publ i shed val ues.
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pH- Dependent Pol ymer Coat i ngs*
Pol ymer Publ i shed
pH Rel ease
Eudragi t 0 S-100 7.0
Eudragi t 0 FS-30D 7.0
Eudragi t 0 L-100 6.0
Cell ul ose acetate pht hal ate 6. 0
Cell ul ose acetate tri mel I i tat e 5. 5
Eudragi t L- 30D- 55 5. 5
Eudragi t 0 L- 100- 55 5.5
Hydroxypr opyl methyl cell ul ose pht hal ate 55 5. 5
Hydroxypr opyl methyl cell ul ose pht hal ate 50 5. 0
Pol yvi I yl acetate pht hal ate 5. 0
*[ Hua, Front Pharmacol 11: Arti cl e 524 (April 2020) 1
For colonic release, colon-targeted drug
delivery systems have been actively pursued because
convent i onal non-targeted therapy can have undesi rabl e
side-effects and low efficacy due to the systemic
absorption of drug before reaching the target site.
Li u et al . , Eur. J. Pharm. Bi opharm. 74: 311-315 ( 2010) ,
adopted dual coati ng approach by usi ng the al kal i ne
aqueous sol uti on of Eudragi t S with bufferi ng agents
for i nner I ayer and the organi c sol uti on of Eudragi te S
for outer I ayer, accel erati ng the drug di ssol uti on at
pH val ues greater than 7. Subsequently, Varum et al
Eur. J. Pharm. Bi opharm. 84: 573-577( 2013) , evaluated in
vivo performance of this dual coated system in humans,
demonst rat i ng more consi stent di si ntegrati on of dual
coated tabl ets mai nl y in the lower intestinal tract.
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CA 03175637 2022- 10- 14
Hashem et al . , Br. J. Pharm. Res. 3: 420-434
( 2013), developed mi cr ospher es combining time-and pH-
dependent systems for col oni c deli very of predni sol one.
By usi ng a combi nation of Eudragi t S and ethyl
cell ul ose, they achi eyed greater col oni c drug delivery,
whi I e prevent i ng premature drug rel ease i n the upper
i nt est i ne.
Eudracol i s another exampl e of a multi - unit
technol ogy provi di ng targeted drug delivery to the
colon, with delayed and uniform drug release. This
system i s based on coati ng the pellet with Eudragi t
RL/ RS and Eudragi t FS 30D, providing colon-specific
drug rel ease in a pH-and time-dependent manner [ Patel ,
Expert Opi n. Drug Del i v. 8:1247-1258 ( 2011) ] .
One composi ti on that targets the small
i nt est i ne comprises a di I uent medi um of sugar/sucrose
beads coated with part i cul ate rose bengal ( RB) that i s
coated with one or a pl ural i ty of layers of a
( met h) acryl ate copol ymer t hat i s composed of about 60
to about 95% by wei ght free radical polymerized C1-C4-
al kyl esters of acryl i c or met hacryl i c aci d and about 5
to about 40% by wei ght ( met h)acryl ate monomers with an
aci di c group i n the al kyl radi cal .
Parti cul arl y suitable (meth)acryl h) acryl ate
copol ymers i ncl ude about 10 to about 30% by wei ght
methyl met hacryl ate, about 50 to about 70% by wei ght
methyl acryl ate and about 5 to about 15% by wei ght
met hacryl i c acid ( Eudragi t FS type). Si mi I ar I y
suitable, are ( met h)acryl ate copolymers of about 20 to
about 40% by wei ght met hacryl i c acid and about 80 to
about 60% by wei ght methyl met hacryl ate ( Eudragi t S
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CA 03175637 2022- 10- 14
type) .
The word "( met h) acryl ate" is used herein to
mean that either or both of acryl ate and met hacryl ate
monomers can be used.
These coati ng polymers permit little if any
HX compound release prior to the particles leaving the
stomach. The pH value of the fluid within the duodenum
typically is about 6 and rises to about 7.4 toward the
i I eum.
A usual tablet or lozenge can be prepared by
admi xt ure of I act ose ( 20%) and act i ve i ngredi ent ( 80%;
HX compound) mixed in a high-speed mixer ( DI OSNA type
P10, Osnabruck, Germany) . An aqueous sol uti on
contai ni ng the exci pi ent pol yvi nyl pyrrol i done ( PVP)
such as povi done ( Si gma- Al dr i ch International GmbH,
Buchs, CH) i s added i n smal I amounts unti I a
homogeneous composition is obtained. The moist powder
mixture is screened. Tablets are subsequently made
therefrom as is well-known, and dried.
The resul ti ng tabl et s or I ozenges are
thereafter preferably coated with a protective pol ymer
film, of ten usi ng fl ui di zed bed equi pment . Fi I m-
f ormi ng pol ymers are normal I y mixed with pl asti ci zers
and release agents by well-known processes. The film
formers can in this case be in the form of a solution
or suspensi on. The exci pi ents for the film formation
can I i kewi se be di ssol ved or suspended.
Organi c or
aqueous sol vents or di spersants can be used.
Stabilizers can be used i n addi ti on to stabilize the
dispersion (for example: Tween 80 or other suitable
emul si f i ers or stabilizers).
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Exampl es of rel ease agents are gl ycerol
monostearate or other suitable fatty acid derivatives,
si I i ci c aci d der i vat i yes or t al c. Exampl es of
pl asti ci zers i ncl ude propyl ene gl ycol , pht hal at es,
pol yet hyl ene gl ycol s, sebacates or ci t rates, and other
substances mentioned above and in the literature.
Another preferred type of medi cament i s a
water- sol ubl e capsul e or bl i ster that contai ns a
pl ural i ty of part i cl es of an HX compound such as rose
bengal di sodi um or rose bengal I act one that are covered
with one or more I ayers of polymeric resi n that rel ease
the HX compound quickly upon dissolution or
di si ntegrati on of the capsul e i n water or body fl ui d.
Capsul es are typi call y made of gel at i n and are often
ref erred to as gel caps.
Gel at in is an ani mal product.
Vegetari an capsul es are often made of hydroxypropyl
methyl cel I ul ose ( HPMC).
I n some embodi ments, the HX compound is
di rect I y layered with one or more coats of the pol ymer
to form particles that are general I y spheri cal i n
shape.
Such particles are often ref erred to as beads.
I n a preferred aspect, part i cl es ( beads) are si zed so
as that about 90 percent by wei ght pass through a 20
mesh si eve ( openi ng = 850 m) screen and about 90
percent by wei ght are retai ned on an 80 mesh si eve
(openi ng = 180 r11) screen.
Exemplary pH value-sensitive coating
pol ymeri c resi ns are di scussed above. The pH val ue-
sensi ti vi ty of coati ng pol ymeri c resi ns is to be
understood i n terms of physi ol ogi call y present pH
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val ues al ong the GI tract such as those di scussed
above.
I n other embodi ments, small pellets such as
sugar/starch seeds, non- parei Is or pri I I s, which are
small , general I y spherically-shaped cores, are coated
with one or a plurality of layers of the HX compound
and one or more layers of polymeric coating.
Illustrative sugar/starch cores are sugar spheres NF
that pass through an about 40 mesh si eve (425 mm
openi ng) screen to an about 50 mesh si eve ( 300 mm
openi ng) screen, that contai n not less than 62.5
percent and not more than 91.5 percent sucrose,
calculated on the dry basis, the remainder consisting
primarily of starch. ( USP NF 1995 2313).
I n an ill ust rat i ve example, a 100 kilogram
( kg) quantity of di sodi um rose bengal , a 7.1 kg
quantity of cross- I i nked car boxymethyl cell ul ose
(preferably croscarmel I ose sodi um NF), and an 11.9 kg
quantity of starch NF, are each di vi ded i n half, and
the three constituents are blended together to form two
i dent i cal batches. Each of the batches is mill ed
through an 80 mesh screen usi ng a mill such as a
Fi tzpat ri ck Mill . The two mill ed batches are then
bl ended to form a mixture, whi ch i s tested for
composi ti on i n accordance with accepted quality
assurance testing methods that are well-known by those
skilled in the art.
The di sodi um rose bengal mi xture i s
subsequently di vi ded i nto three equal parts, with a
f i rst part remai ni ng whol e, and second and t hi rd parts
each divided into lots of 50 percent, 30 percent and 20
percent. A 25.6 kg quantity of 40-50 mesh sugar/starch
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seeds (e. g. , sugar spheres NF) is placed in a stai nl ess
steel coati ng pan. An 80 liter ( L) quantity of 5
percent povi done/ i so- propanol ( I PA) solution is
prepared for sprayi ng onto the part i cl es.
The coati ng pan is started with the sugar
spheres, onto whi ch i s sprayed an appl i cat i on
(approximately 0.173 kg per appl i cat i on) of the
povi done- al cohol solution, and onto which is sifted an
application (approximately 0.32 kg) of the di sodi um
rose bengal mixture from the first part (that part that
remai ned whole). Si fti ng i s done usi ng a standard
sifter. The sprayi ng and si fti ng steps are conti nued
until the f i rst part of the mixture has been appl i ed to
the sugar spheres to form a batch of partially coated
spheres.
The partially coated spheres are then divided
i nto two equal lots, each I ot bei ng pl aced i n a coati ng
pan. Separately for each of the two I ots, sprayi ng of
the povi done/ I PA solution and sifting of the di sodi um
rose bengal mixture as di vi ded i nto the 50 percent I ots
conti nues until the 50 percent lots have been applied
to the spheres. Fol I owi ng appl i cat i on of the 50
percent lots, the spheres can be screened usi ng a 25
mesh screen if necessary.
The sprayi ng of the povi done/ I PA sol uti on and
si fti ng of the di sodi um rose bengal mixture as divided
into the 30 percent lots commences and conti nues unti I
the 30 percent I ots have been appl i ed to the spheres.
The coated spheres can be rescreened usi ng a 25 mesh
screen.
Spraying of the povi done/I PA solution and
si fti ng of the di sodi um rose bengal mixture conti nues
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CA 03175637 2022- 10- 14
usi ng the mixture as di vi ded i nto the 20 percent lots
until the 20 percent lots have been applied to the
spheres. At this poi nt i n the process, the enti re
quantity of the di sodi urn rose bengal mixture has been
appl i ed to the spheres, and about 50 kg of the 5
percent povi done/1 PA sol uti on has been applied to the
spheres.
A 7.5 percent povi done/I PA solution is
prepared and applied to the spheres as a sealant. The
sealed spheres are tumble dried for about one hour,
wei ghed, and pl aced i n an oven at about 122 oF ( 50 0C)
for 24 hours. After dryi ng, the spheres are screened
through a 20 mesh screen and a 38 mesh screen to form
the immediate (quick or fast as compared to del ayed)
rel ease particles.
The above-discussed HX compound-containing
spheres or thei r capsule (or blister) can al so be
coated with a pH value-sensitive enteri c coati ng
pol ymer as di scussed previ ousl y so that once rel eased
in the GI tract, the spheres do not provide thei r
active i ngredi ent, HX compound, to thei r surroundi ngs
unl ess the pH val ue i s at least that of a desi red GI
tract location.
Another way to control the 1 ocat i on of HX
compound rel ease is to further coat the spheres ( HX-
coat ed part i Cl es) discussed above, with a di ssol uti on-
control I i ng coat of pol ymeri c resi n applied to the
surface of the spheres such that the rel ease of the HX
compound from the spheres i s controlled and rel eased
over a 6-10 hour pen i od. The materials used for this
purpose can be, but are not I i mi ted to, ethyl cell ul ose,
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hydroxypropyl methyl-cell ul ose, hydroxypropyl cell ul ose,
methyl cell ul ose, hydroxyet hyl cell ul ose, ni t rocel I ul ose,
car boxymet hyl - cel I ul ose, as well as copolymers of
et hacryl i c acid and met hacryl i c acid ( Eudragi tg), or
any other acrylic acid derivative (Carbopol g, etc.) can
be used.
I n addi ti on, an enteri c coating material can
al so be empl oyed, either si ngul ar I y, or i n combi nat i on
to the above non- pH- sensi t i ve coatings. These
mat er i al s i ncl ude, but are not I i mi t ed to,
hydroxypropyl methyl cell ul ose pht hal ate and the
pht hal ate esters of all the cel I ul ose ethers. I n
addi ti on, pht hal ate esters of the acryl i c acid
der i vat i yes ( Eudragi t ), or cel I ul ose acetate
pht hal ate.
These coati ng mat er i al s can be empl oyed i n
coati ng the surf aces i n an amount of about 1.0 percent
( w/ w) to about 25% ( w/w) . Preferably, these coati ng
materials are present at about 8.0 to about 12.0
percent ( w/w) .
Exci pi ents
Exci pi ents customary i n pharmacy can be
empl oyed i n a manner known per se i n the product i on of
the HX compound- contai ni ng medi cament . These
exci pi ents can be present i n the core or i n the coati ng
agent.
Pol ymers
Pol ymer i c mat er i al s used as adhesi yes i n
hel pi ng to adhere an HX compound to a sugar pri I I or
sphere i s deemed to be an exci pi ent where coati ng
layers of an HX compound are empl oyed.
Ill ust rat i ve of
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CA 03175637 2022- 10- 14
such poi ymers are poi yvi nyl pyrrol i done and poi yvi nyl
alcohol as are other water-sol ubl e, pharmaceuti cal 1 y
acceptable fi I m- f ormi ng pol ymers such as hydroxypropyl
cel 1 ul ose.
Dryers ( non- sti ck agents)
Dryers have the f ol I owi ng properti es: they have 1 arge
specific surface areas, are chemically inert, are free-
fl owi ng and compri se f i ne part i cl es.
Because of these
properti es, they reduce the tack of polymers containing
polar comonomers as functional groups. Examples of
dryers are: al umi na, magnesi um oxi de, kaol i n, t al c,
fumed si I i ca, bar i um sul phate and cell ul ose.
Di si ntegrants
Di si ntegrants are added to oral sol i d dosage
forms to ai d in thei r di saggregati on. Di si ntegrant are
formul ated to cause a rapi d break- up of sol i ds dosage
forms on contact i ng moi st ure. Di si ntegrati on is
typically viewed as the first step in the dissolution
process. Illustrative di si ntegrants include sodi um
croscarmel I ose, an i nternal 1 y cross-1 i nked sodi um
car boxymet hyl cell ul ose, cross- I i nked
pol yvi nyl pyrrol i done ( crospovi done) and sodi urn starch
gl ycol ate.
Release Agents
Exampl es of rel ease agents are: esters of
fatty acids or fatty ami des, al i phat i c, 1 ong- chai n
carboxyl i c aci ds, fatty al cohol s and thei r esters,
montan waxes or paraff i n waxes and metal soaps;
particular mention should be made of glycerol
monostearate, stearyl alcohol, glycerol beheni c acid
ester, cetyl alcohol, pal mi tic acid, carnauba wax,
beeswax, and the I i ke. The usual proporti onate amounts
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CA 03175637 2022- 10- 14
are i n the range from 0.05 percent by wei ght to 5,
preferably 0.1 to 3 percent by wei ght based on the
copol ymer.
Other exci pi ents customary in pharmacy
Menti on shoul d be made here of, for exampl e,
stabi I i zers, col orants, anti oxi dants, wetti ng agents,
pi gments, gl oss agents. They are typi call y used as
processi ng ai ds and are i ntended to ensure a rel i abl e
and reproducible production process and good long-term
storage stability. Further exci pi ents customary i n
pharmacy may be present i n amounts from 0.001% by
wei ght to 10% by weight, preferably 0.1 to 10% by
wei ght, based on t he pol ymer coati ng.
PI asti ci zers
Substances suitable as plasticizers
ordi nari I y have a mol ecul ar wei ght between 100 and
20, 000 and compri se one or more hydrophi I i c groups i n
the molecule, e.g. hydroxyl, ester or amino groups.
Ci t rates, pht hal ates, sebacates, castor oil are
sui table. Exampl es of further sui tabl e pl asti ci zers
are al kyl ci t rates, glycerol esters, al kyl pht hal ates,
al kyl sebacates, sucrose esters, sorbi tan esters,
di butyl sebacate and pol yet hyl ene gl ycol s 4000 to 20
000. Preferred plasticizers are tri butyl citrate,
tri ethyl citrate, acetyl tri ethyl citrate, di butyl
sebacate and di ethyl sebacate. The amounts used are
between 1 and 35, preferably 2 to 10, % by weight,
based on the ( met h) acryl ate copol ymer.
Optimizing Systemic Bi oavai I abi I i ty
The amount of HX compound delivered by a
solid medi cament composition i s substantially the same
as that from an aqueous composition.
Administration of
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CA 03175637 2022- 10- 14
suf f i ci ent RB, as an ill ust rat i ye HX compound, to
achi eve a ci rcul at i ng RB concent rat i on at the I C50
level woul d not by def i ni ti on be toxi c to al I
ci rcul at i ng I eukemi c cel Is ( i . e. , only approximately
half of the I eukemi c cel I s woul d be affected at the
I C50) . I n some embodi ments it can be preferred to
admi ni ster RB i n an amount that is a multi pl e of the
I C50 I evel , up to approxi mate! y 1500 mg ( i . e., 300 mM).
Alternatively, however, it can be suf f i ci ent to kill
only a fraction of tumor cells as a result of an
individual admi ni strati on.
The latter case can be preferable for
avoi di ng a toxi c react i on ( i . e. , "tumor I ysi s
syndrome") that can result from rapidly kill ed tumor
cell burden. Thus, Howard et al . , N Engl J Med
364( 19) : 1844-1854 ( May 12, 2011) report that tumor
I ysi s syndrome is the most common disease-related
emergency encountered by physi ci ans treating
hematol ogi c cancers such as I eukemi a.
As di scussed i n greater detail bel ow, it can
al so be advantageous to kill only a port i on of the
I eukemi c cells dun i ng a si ngl e treatment to i ni ti ate a
f uncti onal i mmune response agai nst remai ni ng I eukemi a
cell burden. A RB- i ni ti at ed functional immune system
response i s believed to occur due at I east i n part from
the act i on of RB-caused necrotic cell debris
ci rcul at i ng i n the body i nduces an i mmune response that
can prol ong the effects of an i ni ti al admi ni strati on of
a hal ogenated xant hene such as RB.
An i nduced i mmune response can take a I onger
ti me to develop than the more i mmedi ate kill i ng of the
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CA 03175637 2022- 10- 14
contacted I eukemi c cell s. That del ay i n effect can
occur because of the ti me needed for i nducti on the
appropri ate B and T cell popul at i ons to attack and kill
the I eukemi c cell s as well as to i nduce I ong I asti ng
memory T cells whose cont i nued ci rcul at i on can protect
the patient from relapse. Such an initial del ay can
be
augmented for the subject's life-time due to the memory
i mmune cell s so i nduced.
Combi nati on Treatment
I n another aspect, an above pharmaceuti cal
composi ti on is used i n conj unct i on with a second,
differently-acting systemic cyt ot oxi c anti-leukemia
agent; i . e. , a cytotoxi c ant i - I eukemi a agent whose
mechanism of act i on i s different from that of the f i rst
cytotoxi c agent, the HX compound. As noted previously,
the hal ogenated xant henes I ocal i ze i n cancer cell
I ysosomes, i ncrease the percentage of cell s in G1 phase
of the cell cycle and i nduces cell death by apoptosi s
[Swift et al . , Oncotargets Ther, 12:1293-1307 (February
2019)].
A first type of second anti-leukemia systemic
cytotoxi c agent i s a so- cal I ed "small mol ecul e. " Such
small mol ecul es can be vi ewed as semi - speci f i c cell ul ar
poi sons i n that they are only general I y more specific
at killing leukemia cells than non- I eukemi c cells.
Al most all small mol ecul e anti cancer agents are I ess
leukemia-specific than a contemplated HX compound, and
can result i n causi ng si ckness, baldness and other
trauma to thei r reci pi ent subj ects that can lead to
subj ects I eavi ng t hei r treatment regi mens.
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CA 03175637 2022- 10- 14
These small mol ecul es typi call y have
molecular weights of about 150 to about 1000 Dal tons
( Da), and preferably about 250 to about 850 Da. Thi s
group of small mol ecul es i nd l udes many of those used to
treati ng hematol ogi c I eukemi as such as cal i cheami ci n
(1368 Da), vi nbl ast i ne ( 811 Da), vi ncri sti ne ( 825 Da),
i mat i nib ( 494 Da), monomet hyl auri st at i n (718 Da),
et oposi de ( 589 Da), daunorubi ci n ( 528 Da), doxorubi ci n
( 544 Da), cl adri bi ne ( 286 Da), fl udarabi ne ( 365 Da),
mi toxantrone ( 444 Da), 6- thi oguani ne ( 167 Da),
met hot rexat e ( 454 Da), 6- mercaptopuri ne ( 152 Da),
azacyt i di ne ( 244 Da), annamyci n (640 Da), soraf eni b
(465 Da), cl of arabi ne ( 304 Da), ci spl at i n ( 300 Da),
i ri not ecan ( 587 Da) and cytabari ne ( 243 Da) . One or
more of the above small molecule anti-leukemia can
comprise a second leukemia cytotoxi c agent. It is
noted that many of these small mol ecul es are used as
thei r sal ts, prodrugs and/or esters, whi ch consequently
have greater molecular weights than those rounded
val ues above.
A pharmaceuti cal composi ti on havi ng a second
systemic cytotoxi c anti-leukemia agent can al so contain
a small mol ecul e as above-described that is conj ugated
to a I ager mol ecul e such as a protei n, detergent and/or
pol ymer such as pol y( ethyl ene gl ycol ) [ PEG] . Such
conj ugati ons of ten mi ni mi ze the toxi city of the small
molecule and enhance si t us of delivery as use of an
anti body that bi nds to a I eukemi c cell . Addi ti
onal I y,
a small mol ecul e cytotoxi c agent can be envel oped
wi t hi n a Ii posome, mi cel I e or cycl odextri n mol ecul e
that can be adapted to bi nd specifically bi nd to
I eukemi c cells and/or be endocytosed by the leukemia
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CA 03175637 2022- 10- 14
cell . Thi s group of encapsul at ed and conj ugated small
molecules i s i ncl uded with the previously di scussed
small mol ecul e group of second systemi c cytotoxi c
agents as t hei r act i ve cytotoxi c agent i s a small
mol ecul e.
Ill ust rat i ve of such second systemic
anti I eukemi a cytotoxi c agents are I i posomal
daunorubi ci n, I i posomal annamyci n, sphi ngosomal
vi ncri sti ne, I i posomal cytarabi ne, a cal i cheami ci n-
conj ugated CD33 anti body cal led gemt uzumab ozogami ci n
and a chi mer of CD30 anti body and monomethyl auri stati n
E cal I ed brent uxi mab vedoti n.
Briefly, I i posomes are general I y spheri cal I y-
shaped artificial vesi cl es typi cal I y prepared from
chol est erol and phosphol i pi d mol ecul es that consti tute
one or two bi layers and encapsul ate the smal I mol ecul e
second systemi c cytotoxi c agent to assi st deli very.
See, Akbarzadeh et al . , Nanoscal e Res Lett, 8: 102
(2013) .
Cal i cheami ci n, i s a hi gh mol ecul ar wei ght
small mol ecul e ( 1368 Da), and contai ns four I i nked
sacchari des i nt err upted by a benzothi oate S- ester
I i nkage as well as an ene- di yne group that cleaves DNA
sequences. Cal i cheami ci n is too toxi c to be used
al one, LD50 i n nude mi ce of 320 [ig/ kg [ Di J oseph et al
BI ood 103: 1807- 1814 ( 2004) ] . Si mi I ar I y,
monomethyl
auri stati n exhi bits general (broad range), hi gh
toxi city [ I C50 < 1 nM for several cancer cell I i nes;
ApexBi o Technology Product Catalog ( 2013) ] that is
mediated by linkage to an anti body against CD30 ( a TNF
receptor-family member that is a cell membrane protei n
- 48 -
CA 03175637 2022- 10- 14
and cancer marker) was reported useful agai nst I arge
cell I ymphoma and Hodgki n' s di sease [ Franci sco et al
Blood 102: 1458- 1465 ( 2003) ], whereas linkage to an
anti -CD79b monocl onal provi ded an advantage in treati ng
three xenograft models of NHL [ Dornan et al . , Blood
114: 2721- 2729 (2009)1.
A systemic anti-leukemia medi cat i on that is a
small molecule ( non- protei naceous, less than about 1000
grams/ mole) or a I arger protei naceous molecule, i s
admi ni stered to the subj ect mammal to be treated such
that the medication spreads throughout the subject's
body. I nt ravenous admi ni strati on is one preferred
method to achieve that spread of medication. On the
other hand, i mat i ni b i s usual I y admi ni stered oral I y.
Illustrative small molecule anti-cancer
medi cat i ons useful for treating I eukemi a include
doxorubi ci n, et oposi de, vi ncri sti ne, ci spl at i n,
i ri not ecan and cytarabi ne were used i n parental
application Seri al No. 16/ 688, 319, whereas an exemplary
protei naceous mol ecul e i s egasparagi nase. Of those
small molecule medications, doxorubi ci n, et oposi de and
vi ncri sti ne each of whi ch woul d be admi ni stered IV to a
mammal i an subj ect appeared to synergi ze i n treatment
with a sub-lethal dose of PV- 10 aqueous RB di sodi um
sol uti on, and are preferred.
It is to be understood that admi ni strati on of
any of the second I eukemi a systemi c cytotoxi c agents
di scussed herei n can be undertaken multi pl e ti mes.
Such multi pl e admi ni strati ons are within the purvi ew of
the treati ng physi ci an, and can be made i n conj uncti on
with an admi ni strati on of the HX compound f i rst
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CA 03175637 2022- 10- 14
leukemia cytotoxi c agent or can be carried out
separate! y.
A useful effective dosage of a small molecule
systemic anti-leukemia medication is the dosage set out
in the label i ng i nf ormat i on of a FDA-, nat i onal - or
i nt er nat i onal agency- approved medi cat i on. Typi cal
I y,
monot herapy dose schedul es are set by determi ni ng the
maxi mum tol erated dose ( MTD) i n early- stage cl i ni cal
trials.
The MTD ( or a close variation thereon) is then
promul gat ed to later-stage cl i ni cal trials for
assessment eff i cacy and more detail ed assessment of
safety. These MTDs frequently become the established
t herapeut i c dose upon compl et i on of cl i ni cal test i ng.
However, because the small mol ecul e, systemi c anti -
leukemia medi cat i on i s contempl at ed for use with an HX
compound i n a sol i d or I i qui d f ormul at i on, a MTD i s the
maximal amount that would normally be used, and that
amount is to be t i t rated downward f ol I owi ng usual
procedures.
Exempl ary dosi ng schedul es for several
systemic anti-cancer ( ant i - I eukemi a) medications
( agent s) t hat can be combi ned wit h hal ogenat ed xant hene
therapy in the present invention are provided in Table
A, below. It is noted that several of the medications
I i sted bel ow are "smal I mol ecul es" as def i ned above,
whereas others are large, protei naceous mol ecul es such
as anti bodi es, preferably monoclonal anti bodi es,
i nhi bit i nf I ammatory chemoki ne act i vi ty. They are
nonet hel ess admi ni stered systemically.
The medi cat i ons
of Table A are usually used as si ngl e active agents.
However, one or more can al so be used together,
parti cul arl y the anti bodi es, as is the case with the
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CA 03175637 2022- 10- 14
immune checkpoi nt i nhi bi tor antibodies that are
di scussed herei naf ter.
Tabl e A
Exempl ary systemi c i mmunomodul at ory
or targeted anti cancer agents
Systemic Agent Typical Dose Schedule
adal i mumab 80 mg i ni ti al dose followed
i n 1 week by 40 mg every
other week SQ
brodal umab 210 mg subcutaneously (SC) at
Weeks 0, 1, and 2, then 210
mg SC q2wk
certol i zumab 400 mg initially and at weeks
pegol 2 and 4 followed by 200 mg
every other week or 400 mg Q4
weeks mai ntenance SQ
etanercept 50 mg twi ce weekly for 3
months followed by 50 mg once
weekly SQ
gol i mumab 50 mg once a month SQ
gusel kumab 100 mg subcutaneous i nj ecti on
once every 8 weeks, after
starter doses at weeks 0 and
4
i nf I i xi mab 5 mg/ kg given as an IV
i nduct i on regi men at 0, 2,
and 6 weeks followed by a
mai ntenance regi men of 5
mg/ kg every 8 weeks
thereafter
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i xeki zumab 160 mg initial dose followed
Q2 weeks with 80 mg unti I
week 12 then 80 mg Q4 weeks
SQ
sari I umab 200 mg every 2 weeks as a
subcutaneous i nj ect i on
secuki numab 300 mg every week for 4 weeks
then 300 mg every 4 weeks SQ
ust eki numab Less than 100 kg: 45 mg
i ni ti ally, week 4 followed by
45 mg every 12 weeks SQ
More than 100 kg: 90 mg
i ni ti ally, week 4 followed by
90 mg every 12 weeks SQ
apremi last Ti t rat ed dose over 5 days to
work up to 30 mg twice daily
PO
met hot r exat e Weekly single oral, IM or IV
to 25 mg per week or
di vi ded 2. 5 mg dose at 12
hour i nterval s for three
doses
cycl ospori ne I ni ti al dose 2.5 mg/ kg/ day
taken twice dai I y as divided
( BI D); dose t i t rated up to 4
mg/ kg/ day BID if response and
I aboratory abnormal i ti es
don' t ensue.
azat hi opri ne Used off label for ski n
diseases, 1.0 mg/ kg oral or
I V as a si ngl e dose or twi ce
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CA 03175637 2022- 10- 14
a day, dose maxi mum i s 2. 5
mg/ kg/ day.
Because of additive or synergistic effects,
the combi nation therapy and method of treatment of the
present i nventi on general I y permit use of the systemi c
agent at a I evel at or bel ow the typi cal dose schedul e
for the systemi c agent, such as those descri bed i n
Table A, when used with an IV admi ni strati on therapy,
such as that descri bed bel ow. However, the dosi ng
schedules provided in Table A provide a useful guide
for begi nni ng treatment from which dosages can be
ti t rat ed to lessened amounts as seen appropriate by the
physician caring for a given patient.
It is noted that a HX compound and a second
cytotoxi c anti - I eukemi a agent need not be admi ni stered
together nor by the same means of admi ni strati on.
Thus, a pi I I or capsul e form can be used for can be
used to admi ni ster the HX compound f i rst cytotoxi c
anti -1 eukemi a agent, whereas the small or 1 arge
molecule second anti -1 eukemi a agent i s admi ni stered by
IV or orally, like i mat i nib.
Those skilled in the art
are aware of the van i ous met hods of admi ni steri ng anti -
1 eukemi a agents.
A second type of second systemic cytotoxi c
agent usef ul for combi nat i on t r eat ment wi th a
hal ogenated xanthene such as that present i n aqueous RB
di sodi um sol uti on or a before-described solid dosage
form i s an i mmune checkpoi nt i nhi bi tor, that can al so
be viewed as a special systemic anti-leukemia
medi cat i on. An i mmune checkpoi nt i nhi bi tor is a drug
that bi nds to and bl ocks certai n checkpoi nt protei ns
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CA 03175637 2022- 10- 14
made by i mmune system cell s such as T cell s and some
I eukemi a cell s. When not bl ocked, those protei ns
i nhi bit i mmune responses, hel pi ng keep i mmune responses
i n check and keepi ng T cell s or other immune cell s from
kill i ng leukemia cells. BI ocki ng those immune
checkpoi nt protei ns rel eases the "brakes" on the i mmune
system permitting immune cells to become activated and
kill I eukemi a cell s.
A useful i mmune checkpoi nt i nhi bi tor i s
preferably a human or humani zed monocl onal anti body or
bi ndi ng portion thereof whose admi ni strati on blocks the
act i on of those certai n protei ns. That blockage
permits the immune system to recognize the leukemia
cell s as foreign and assi St i n el i mi nati ng those
I eukemi a cell s from the body.
Ill ust rat i ve i mmune checkpoi nt inhibitors
include the ant i - CTLA- 4 ( cyt ot oxi c T I ymphocyt e-
associ at ed anti gen 4) monocl onal anti bodi es i pi I i mumab
and t remel i mumab that are desi gned to counter down-
regulation of the immune system by blocking CTLA- 4
activity and thus augment T cel I response agai nst
I eukemi a.
Si mi I ar I y, monocl onal anti bodi es such as
pi di I i zumab, ni vol umab, ti sl el i zumab, spart al i zumab,
cemi pl i mab, pembrol i zumab, camr el i zumab, si nti I i mab,
tor i pal i mab, and dostar I i mab bi nd to PD-1 ( programmed
death 1) receptor to counter down- regul at i on of the
i mmune system and augment T cel I responses to cancerous
cells.
Three monocl onal antibodies that target the
immune checkpoi nt protein I i gand (anti - PD- L1) for the
PD-1 receptor ( ant i - PD- 1) are at ezol i zumab, avel umab,
and durval umab. I ni ti al work with anti bodi es to the
PD-1 receptor I i gands, PD- Li and PD- L2, such as BMS-
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CA 03175637 2022- 10- 14
936559 and MEDI 4736 ( durval umab) to PD-Li, al so
i ndi cat e i nhi bi ti on of down- regul at i on of the i mmune
system and an augmented T cell response agai nst
I eukemi a.
The above i mmune checkpoi nt i nhi bi tor
anti bodi es have been found useful when admi ni stered
si ngl y al ong with a HX compound as well as usi ng two
different types of i mmune checkpoi nt i nhi bi tors al ong
with an HX compound. A poster by Patel et al
AMERICAN SOCIETY of CLINICAL ONCOLOGY ( ASCO) 2020
VI RTUAL SCI ENTI Fl C PROGRAM May 29- 31, 2020 provi ded
data from a study that utilized rose bengal di sodi um
that was i nj ected i nt rat umor al I y i nt o uveal mel anoma
tumors metastati c to the liver al ong with either a
systematic admi ni strati on of anti - PD-i, or systemi c
admi ni strati ons of both anti - PD- 1 and CTLA- 4
anti bodi es.
More recently, several further groups of
anti bodi es with checkpoi nt i nhi bi tor act i vi ty have been
identified, and because of t hei r similarity of act i on,
are deemed herei n to be i mmune checkpoi nt i nhi bi tors.
One ill ust rat i ve group i mmunoreact with the cell
surf ace receptor 0X40 (CD134) to stimulate
prol i f erat i on of memory and effector T- I ymphocytes, and
thereby st i mul ate a T- cel I - medi at ed immune response
agai nst cancerous cel I s. Exempl ary such humani zed
anti - 0X40 monocl onal anti bodi es i ncl ude those presently
referred to i n the literature as gsk3174998 ( I gG1),
pogal i zumab (MOXR0916), MED10562 and the human ant i -
OX40 I gG2 antibody designated PF- 04518600 ( PF- 8600) .
Another group i mmunoreacts with lymphocyte
act i vat i on gene 3 prot ei n ( ant i - LAG- 3; CD223) t hat
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CA 03175637 2022- 10- 14
negatively regul at es T lymphocytes by bi ndi ng to the
ext race! I ul ar domai n of the I i gand, thus avoi di ng
autoi mmuni ty caused by T cell overact i vat i on.
LAG-3 is
an i mport ant i mmune checkpoi nt in vi vo and pl ays a
bal anced regul at ory rol e in the human i mmune system
[ Shan et al . , Oncol Lett 20: 207 ( 2020)1.
The molecule LAG-3 blocks the signal
transduct i on pathway of T cell act i vat i on; however, the
intracellular segment of the LAG-3 molecule produces
i mmunosuppressi ve signals, which have been found to
regul ate CD4+T cell activity.
LAG-3 regul at es the
immune response of T cel Is in three ways: Fi rst, it
di rect I y i nhi bits the proliferation and activation of T
cell s vi a negative regul at i on of T cells. Second,
it
can promote the i nhi bi tory function of regul at ory T
cells (Tregs), and the T cel I response can then be
indirectly inhibited.
Thi rd, it can prevent T cell
activation by regul at i ng the function of antigen
presenting cells ( APCs) [ J ol I er et al . , Curr Top
Mi crobi ol I mmunol 410: 127-156 ( 2017) ] .
To date, no monocl onal anti body to LAG-3 is
known to be been approved for sale and use by the US
Food and Drug Admi ni strati on.
Studies are ongoi ng
usi ng a humani zed I gG4 monocl onal anti body from Merck
ref erred to as MK-4280, and another from Bristol Myers
Squi bb with the I NN name rel at I i mab.
A still further type of immune checkpoi nt
i nhi bi tor i s a monocl onal anti body agai nst CD47 and
macrophage checkpoi nt i nhi bi tor that i nterf eres with
recognition of CD47 by the SIRPa receptor on
macrophages, thus bl ocki ng the "don' t eat me" signal
used by cancer cel Is to avoi d bei ng i ngested by
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CA 03175637 2022- 10- 14
macrophages. Thi s monocl onal , whose I NN name i s
magrol i mab, i s bei ng devel oped by Gil i ad Sci ences, I nc.
i n several hematol ogi c and sol i d tumor mal i gnanci es,
i ncl udi ng myel odyspl ast i c syndrome ( MDS) .
Magrol i mab
has been granted Fast Track Desi gnat i on by the FDA for
the treatment of myel odyspl ast i c syndrome ( MDS), acute
myel oi d I eukemi a ( AML), diffuse I arge B- cell I ymphoma
( DLBCL) and f ol I i cul ar I ymphoma.
Magrol i mab has al so
been granted Orphan Drug Designation by the FDA for MDS
and AML and by the European Medicines Agency for AML.
Monocl onal anti bodies to T cell
i mmunogl obul in and muci n domain 3 (anti -TI M-3), another
checkpoi nt marker, are i n early devel opment by Novart i s
Oncol ogy Co. under the I NN name sabatol i mab ( earl i er
MBG- 453) for use i n MDS and AML therapy based on
di f f erent i al I eukemi c stem cell expressi on, its role as
a co- i nhi bi tory T- cell co- receptor, and possi bl y a rol e
in promot i ng anti body-dependent cellular phagocyt osi s
(ADCP) . TI M- 3 is expressed on AML leukemic progenitors
but i s not seen on normal hematopoi et i c stem cell s and
its expression has been correlated with severity of
myel odyspl ast i c syndromes as well as the I i kel i hood of
progression to AML. There are currently multiple
trials of TI M-3 anti body MBG- 453 in f ront I i ne
myel odyspl ast i c syndromes and AML, with encouraging
ant i I eukemi c activity presented when used in
combi nat i on with deci tabi ne.
I nt act monocl onal anti bodi es, as well t hei r
paratope- contai ni ng port i ons (binding site-containing
port i ons) such as Fab, Fab' , F( ab' )2 and Fv regi ons, as
well as si ngl e- stranded pept i de bi ndi ng sequences can
be useful as i mmune checkpoi nt protei n i nhi bi tors.
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Most of these anti bodi es are admi ni stered vi a an IV
route. I nt act checkpoi nt i nhi bi ti ng monocl onal
anti bodi es have half-lives i n a human body of about one
to three weeks [ e. g. , Yervoye ( i pi I i mumab) t ermi nal
t 1/ 2 = 15.4 days; package insert 12/2013; Keyt ruda()
( pembrol i zumab) termi nal t 1/ 2 = 23 days; package i nsert
03/201]], and single-stranded ol i go or pol ypept i des
tend to have shorter hal f - I i ves in vivo.
Because of the relatively short half-lives of
the smal I mol ecul e second cytotoxi c anti - leukemia
agents and a hal ogenated xanthene compound- contai ni ng
medi cament, both medi caments can be admi ni stered i n a
si ngl e composition or i n separate composi ti ons. If
admi ni stered separately, it is preferred to admi ni ster
both types of anti - cancer (anti - leukemia) agent wi t hi n
mi nut es to about 3 hours of each other. More
preferably, both are admi ni stered wi t hi n less than one
hour of the other.
The word ''admi ni strati on" i s used herei n to
mean the beginning of a treatment regimen. Thus,
swal I owi ng a tablet or other per os dosage form i s the
begi nni ng of a treatment regi men, as i s the ti me at
which an IV fl ow is begun. When both first and second
cytotoxi c anti -I eukemi a agents are present together i n
the same, si ngl e composi ti on, admi ni strati on begi ns
when that unitary composi ti on enters the subject's
body.
Where the second cytotoxi c systemi c anti -
1 eukemi a agent i s an i mmune checkpoi nt i nhi bi tor such
as a monocl onal anti body, the hal ogenated xant hene
compound and the second cytotoxi c anti-leukemia agent
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CA 03175637 2022- 10- 14
i mmune checkpoi nt i nhi bi tor can be admi ni stered
together or one before the other, with the second
cytotoxi c anti I eukemi a agent i mmune checkpoi nt
i nhi bi tor bei ng admi ni stered up to about one month
prior to the hal ogenated xanthene.
Preferably, the two
cytotoxi c ant i - I eukemi a agents are admi ni stered
together or with the second systemic cytotoxi c anti -
I eukemi a agent i mmune checkpoi nt i nhi bi tor bei ng
admi ni stered wi t hi n a few days after the hal ogenated
xant hene. A second systemic cyt ot oxi c anti-leukemia
agent i mmune checkpoi nt i nhi bi tor can al so be
admi ni stered about one month after the hal ogenated
xant hene.
Studi es
Mi ce:
Female C17 SCI D mice from Charles River
Cell I i ne used in xenograf t :
The SEM cel I I i ne was i ni ti ally established from a 5
year old female with B acute I ymphobl ast i c leukemia.
It carries the MLL-AFFF1 gene fusion and heterozygous
for CDKN2A and TP53.
The utility of this cell I i ne i n
anti -cancer drug sensitivity studies has been descri bed
( Bar ret i na et al . Nature 483: 603- 607, 2012) .
2.5x106 exponentially growing SEM cells
[ I abel led with green fl uorescent protei n (GFP) ] were
injected i nt ravenous! y i nto each ani mal and the
establishment of tumors was monitored. After 4 weeks
to permit the growth of the tumors, mice were
randomized to three groups.
Group 1 ( n=9). Control :
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These ani mal s recei ved 100 ilL of PBS given oral I y twi ce
a week for two weeks.
Group 2 ( n=8). Treatment Cohort I :
These animals received 25 1.11_ of PV- 10 (bob rose bengal
di sodi um w/v i n 0. 9 percent i n aqueous sal i ne) di I uted
i n PBS to a f i nal vol ume of 100 111_ and given oral I y by
gavage twi ce a week for 2 weeks.
Group 3 ( n=8). Treatment Cohort I I .
These animals received 12.5 tiL of PV- 100 ( as discussed
above) di I uted i n PBS to a final vol ume of 100 !IL and
given oral I y by gavage twi ce a week for 2 weeks.
Evi dence of di sease progressi on was moni tored
i n al I ani mal s and survi val was f ol I owed up to 120 days
following the i ni ti at i on of treatment. Data are
presented i n Fig. 1 as Kapl an- Mei er esti mates.
As is seen from the graphs of Fig. 1, oral
delivery of the HX compound is clearly evidenced by
dose-dependent survival of the treated mice.
The results di scl osed i n parental appl i cat i on
Seri al No. 16/688, 319 showed that el even commerci ally
avail abl e I eukemi a cell I i nes der i ved from pat i ents
with either pri mary or relapsed pedi at ric I eukemi a that
were treated with PV- 100 and two primary leukemia
samples in cell culture could be killed by
admi ni strati on of i ncreasi ng doses of PV- 10 . Cell
vi abi I i ty was measured by al amar bl ue assay, 96 hours
post- t r eat ment .
PV- 100 admi ni strati on decreased I eukemi a cell
vi abi I i ty i n a concent rat i on and ti me dependent manner
in the el even pediatric leukemia cell lines ( mean 1050
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92.8 M), and three primary leukemia samples ( mean I C50
122. 5 M) exami ned. The resul ts show that PV- 10 i s
cytotoxi c to I eukemi a cell I i nes with a mean I C50 val ue
of 92.8 124 (Table 1, below) and is cytotoxic to two
primary leukemia samples with a mean I C50 value of
122.5 p.M (Table 2, below) .
Table 1*
Cell Line Cell Type PV-10 I C50
PM
KOPN8 I nf ant ALL 150
SUPB15 B- ALL 129
CEM- Cl T- ALL 121
TI B-202 AML 118
SEM B- ALL 99
CCRF- SB B- ALL 88
Kas umi 1 AML 72
MV4- 11 Bi phenot ypi c 68
Mol m13 AML 42
Mol t 4 1-ALL 41
Mol t 3 1-ALL 35
Mean 92.8
*Hal f maxi mal i nhi bi tory concentration (1 C50)
val ues f or pedi at ri c I eukemi a cel I I i nes treated wi th PV- 10
for 96 hours.
Tabl e 2**
Cell Type PV-10 1050 pm
1-ALL 150
Infant AML 95
Mean 122.5
**Hal f maxi mal i nhi bi t ory concent r at i on ( I C50)
val ues for pri nary pedi at ri c I eukemi a sampl es treated
with PV- 10 for 96 hours.
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Si mi I ar resul ts were separately obtai ned
usi ng I eukemi a cel I I i nes CCRF- CEM, HL- 60( TB) , K- 562,
MOLT-4, RPM! - 8226 and SR.
It was surpri si ng that I eukemi a cell s coul d
be killed at all at the concentrations of RB di sodi urn
used i n these studi es, which come out to approximately
10- 4 to about 10- 5 molar based on the I C50 values.
Even so, the concent rat i ons of HX compound requi red
were greater than those bel i eyed achi eyed by oral
admi ni strati on i n the present studies as was discussed
previ ousl y. Thus, it was st i I I more surpri sing that
62.5 % of the HX Compound-treated leukemic mice
survived for 120 days whereas the untreated leukemic
mice and those treated with lesser amounts of HX
compound exhi bi ted much poorer survi val rates as are
shown by the data i n Fi g. 1.
Each of the patents, patent applications and
art i cl es cited herei n is i ncorporated by reference.
The arti cl es "a" and "an" are used herei n to refer to
one or to more than one ( i . e., to at I east one) of the
grammatical object of the article. Each of the
patents, patent appl i cat i ons and art i cl es cited herei n
i s i ncorporated by reference.
The f oregoi ng descri pti on and the exampl es
are intended as illustrative and are not to be taken as
I i mi ti ng. St i I I other van i at i ons within the spi ri
t and
scope of t hi s i nventi on are possi bl e and will readily
present themselves to those ski I I ed i n the art.
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